DNA encoding a novel human inhibitor-of-apoptosis protein

ABSTRACT

The present invention relates to novel human inhibitor-of-apoptosis polypeptides, designated HIAP3, polynucleotides encoding the polypeptides, methods for producing the polypeptides, expression vectors and genetically engineered host cells for expression of the polypeptides. The invention further relates to methods for utilizing the polynucleotides and polypeptides in research, diagnosis, and therapeutic applications.

FIELD OF THE INVENTION

[0001] This invention relates, in part, to newly identifiedpolynucleotides and polypeptides; variants and derivatives of thepolynucleotides and polypeptides; methods of making the polynucleotidesand polypeptides, and their variants and derivatives; and uses of thepolynucleotides, polypeptides, variants, and derivatives. In particular,in these and in other regards, the invention relates to novel humaninhibitor-of-apoptosis polypeptides and the polynucleotides which encodethese polypeptides.

BACKGROUND OF THE INVENTION

[0002] Apoptosis, also known as programmed cell death, is a geneticallycontrolled process which plays an important role in development and incellular and tissue homeostasis (Hengartner, M., Exp. Gerontol.32:363-374, 1997; Hoeppner et al., Biochem. Biophys. Acta 1242:217-220,1996; Ellis et al., Annual Rev. Cell Biol. 7:663-698, 1991). Apoptosispermits the elimination of cells which either have been overproduced,developed improperly or have undergone genetic damage and represents amajor host defense mechanism for limiting the replication of infectiveviruses. In contrast to necrotic cell death, which is usuallyaccompanied by swelling and disruption of cellular membranes andinflammation of adjacent tissue, apoptosis is marked by cell shrinkage,blebbing, chromatin condensation, DNA fragmentation and formation ofapoptotic bodies (MacLellan and Schneider, Circ. Res. 81:137-144, 1997;Cohen, G., Biochem. J. 326 (Pt 1):1-16, 1997). Apoptotic cells are thenphagocytosed by neighboring scavenger cells without eliciting aninflammatory response (Wu and Horvitz, Nature 392:501-504, 1998).

[0003] Deregulation of apoptosis has been implicated in the pathogenesisof a variety of diseases. Impaired apoptosis can play a role in cancer(Pan et al., Cancer Surv. 29:305-327, 1997; Thompson, C., Science267:1456-1462, 1995) or chronic viral infection (Clem et al., Science254:1388-1390, 1991; Clem and Miller, Mol. Cell. Biol. 14:5212-5222,1994). Inappropriate (or premature) apoptosis may contribute toneurodegenerative disorders (Roy et al., Cell 80:167-178, 1995; Raff etal., Science 262:695-700, 1996) or acquired immunodeficiency disease(Banda, N., J. Exp. Med. 176:1099-1106, 1992). Premature apoptosis isalso recognized as a contributing cause of myocyte loss inischemia/repurfusion injury, myocardial infarction (MacLellan andSchneider, Circ. Res. 81:137-144, 1997), and congestive heart failure(Feuerstein, G., Trends Cardiovas. Med. 7:249-255, 1997).

[0004] The presence of a novel class of apoptosis inhibitors, known asinhibitor of apoptosis proteins (IAPs) has been reported in theliterature (Liston et al., Apoptosis 2:423-441, 1997). The first IAP wasdiscovered in baculovirus (Crook et al., J. Vir. 67:2166-2174, 1993) andIAPs have now been reported in Drosophila, chick, mouse and human (Hayet al., Cell 83:1253-1262, 1995; Liston et al., supra). Five human IAPshave been identified: HIAP1, HIAP2, XIAP (X-chromosome linked IAP), NIAP(neuronal IAP) and survivin (Ambrosini et al., Nat Med. 3:917-921, 1997;Duckett et al., Embo J. 15:2685-2694, 1996).

[0005] IAPs are a highly evolutionarily conserved family of proteins,containing a number of common structural features (domains). Among theseare an N-terminal domain containing one or more repeats of a domainreferred to as the BIR (baculovirus IAP repeat) domain (Liston et al.,supra), and a C-terminal RING zinc finger domain. These domains arepresent to varying degrees within the known members of the IAP family;HIAP1 and HIAP2 contain three BIR domains and a C-terminal RING domain,while survivin contains only a single BIR domain and no RING domain.

[0006] While the physiological role of IAPs is not exactly clear, somemembers of the IAP family appear to play a regulatory role in apoptosis.Recombinant IAPs were found to suppress apoptosis induced by a varietyof stimuli in different cell types. Drosophila IAPs (DIAP1 and DIAP2)were found to interact with a Decapentaplegic (Dpp) type I receptor,suggesting that these DIAPs may act as negative regulators of the Dppsignaling pathway, which normally leads to cell apoptosis. XIAP, HIAP1and HIAP2 can directly inhibit specific caspases (cysteine containingaspartate specific proteases), enzymes which are involved in thepathways which control apoptosis, and thereby suppress apoptosis(Thornberry, N., Br. Med. Bull. 53:478-490, 1997). However, NIAP wasfound not to inhibit caspases, suggesting that different IAPs may havedifferent mechanisms of action.

[0007] By helping in the regulation of programmed cell death, IAPs playan important role in the maintenance of the appropriate life cycle ofthe various cells of an organism. It is likely that variance from normallevels (either overabundance or deficiency) of IAPs within the cellularenvironment may lead to conditions in vivo which are related to variousdisease states.

[0008] IAPs may play a role in tumor formation. Up-regulated chicken IAPand concommitant suppression of apoptosis were found in chicken cellstransformed by the oncoprotein v-rel, a member of the Rel/NF_(kappa)Bfamily (You et al., Mol. Cell. Biol. 17:7328-7341, 1997). Similarly,baculovirus protein p35 (a baculovirus IAP) is capable of promoting thetransformation of mouse embryo fibroblasts in the presence of theinsulin-like growth factor I receptor (Resnicoff et al., J. Biol. Chem.273:10376-10380, 1998).

[0009] Survivin is undetectable in terminally differentiated adulttissues but expressed in all common human cancers, further suggestingthat apoptosis inhibition may be a general feature of neoplasia(Ambrosini et al., supra).

[0010] Deletion mutations in human NIAP have been linked toinappropriate depletion of motor neurons associated with spinal muscularatrophy, an autosomal neurodegenerative disorder (Xu et al., J. Comp.Neurol. 382:247-259, 1997). In a rat ischemia model, in vivooverexpression of NIAP reduced ischemic damage in the rat hippocampus(Roy et al., supra), indicating that the presence of increased levels ofIAPs could prevent the unwanted cell death characteristic of ischemiaand that elevating the neuronal levels of this IAP may be useful intreating stroke.

[0011] Finally, Stellar and his colleagues were able to block retinalcell death and show significant retention of visual function inDrosophila which exhibited retinitus pigmentosa, a cause of blindness inhumans, by eye-specific expression of the antiapoptotic protein p35(Davidson and Stellar, Nature 391:587-591, 1998).

[0012] These data suggest that antiapoptotic proteins, such as IAPs, aregood candidates for use in the therapeutic intervention of diseasescaused by altered apoptosis.

[0013] There is a need, therefore, for identification andcharacterization of proteins that influence apoptosis. In particular,there is a need to isolate and characterize additional IAPs, akin toknown IAPs, which may be employed, therefore, for ameliorating orcorrecting dysfunctions or disease associated with inappropriateapoptosis; in cancer and chronic viral infections, where IAPs may beoverproduced, as well as in neurodegenerative disorders, chronic heartfailure and dysfunctional immune response, where a deficiency in IAPsmay exist.

SUMMARY OF THE INVENTION

[0014] The present invention provides a polynucleotide sequence whichuniquely encodes a novel human inhibitor-of-apoptosis protein.Designated HIAP3, the polypeptide is characterized by structuralfeatures common to the inhibitor-of-apoptosis protein family, such asBIR and RING domains. The polynucleotide sequence, designated in lowercase, hiap3, and described in FIG. 1, encodes the amino acid sequence,which is designated HIAP3, and is shown in FIG. 2.

[0015] Toward these ends, and others, it is an object of the presentinvention to provide polypeptides, inter alia, that have been identifiedas a novel HIAP3 by homology between the amino acid sequence set out inFIG. 2 and known amino acid sequences of other IAP proteins.

[0016] It is a further object of the invention, moreover, to providepolynucleotides that encode HIAP3, particularly polynucleotides thatencode the polypeptide herein designated HIAP3.

[0017] In accordance with this aspect of the invention there areprovided isolated polynucleotides encoding HIAP3, including mRNAs,cDNAs, genomic DNAs and, in further embodiments of this aspect of theinvention, biologically, diagnostically, clinically or therapeuticallyuseful variants, analogs or derivatives thereof, or fragments thereof,including fragments of the variants, analogs and derivatives.

[0018] Among the particularly preferred embodiments of this aspect ofthe invention are naturally occurring allelic variants of hiap3polynucleotides.

[0019] It also is an object of the invention to provide HIAP3polypeptides that may be employed to treat neoplasia, neurodegenerativedisorders, immune disorders, chronic viral infections, or chronic heartfailure.

[0020] In accordance with this aspect of the invention there areprovided novel polypeptides of human origin referred to herein as HIAP3as well as biologically, diagnostically or therapeutically usefulfragments, variants and derivatives thereof, variants and derivatives ofthe fragments, and analogs of the foregoing.

[0021] Among the particularly preferred embodiments of this aspect ofthe invention are variants of HIAP3 encoded by naturally occurringallelic variants of the hiap3 polynucleotide.

[0022] It is another object of the invention to provide a method ofproducing the aforementioned polypeptides, polypeptide fragments,variants and derivatives, fragments of the variants and derivatives, andanalogs of the foregoing. In a preferred embodiment of this aspect ofthe invention there are provided methods of producing the aforementionedHIAP3 polypeptides comprising culturing host cells having expressiblyincorporated therein an exogenously-derived HIAP3-encodingpolynucleotide under conditions for expression of human HIAP3 in thehost and then recovering the expressed polypeptide.

[0023] In accordance with another object of the invention there areprovided products, compositions, processes and methods that utilize theaforementioned polypeptides and polynucleotides for research,biological, clinical and therapeutic purposes, inter alia.

[0024] In accordance with certain preferred embodiments of this aspectof the invention, there are provided products, compositions and methods,inter alia, for, among other things: assessing HIAP3 expression in cellsby determining HIAP3 polypeptides or HIAP3-encoding mRNA; assayinggenetic variation and aberrations, such as defects, in hiap3 genes; andadministering an HIAP3 polypeptide or polynucleotide encoding HIAP3 toan organism to alter the level of HIAP3 activity.

[0025] In accordance with certain preferred embodiments of this andother aspects of the invention there are provided probes that hybridizeto hiap3 sequences.

[0026] In certain additional preferred embodiments of this aspect of theinvention there are provided antibodies against HIAP3 polypeptides. Incertain particularly preferred embodiments in this regard, theantibodies are highly selective for HIAP3, and may be employeddiagnostically to detect increased HIAP3 expression, which may beassociated with conditions in which inhibition of apoptosis is toostrong, such as cancer.

[0027] In a further aspect of the invention there are providedcompositions comprising an hiap3 polynucleotide or an HIAP3 polypeptidefor administration to cells in vitro, to cells ex vivo and to cells invivo, or to a multicellular organism. In certain particularly preferredembodiments of this aspect of the invention, the compositions comprisean hiap3 polynucleotide for expression of an HIAP3 polypeptide in a hostorganism for treatment of disease. Particularly preferred in this regardis expression in a human patient for treatment of a disease state whichis alleviated by increasing the level of HIAP3 activity.

[0028] In a further aspect of the invention there are provided ribozymesand polynucleotides complementary to hiap3 polynucleotides (i.e.antisense polynucleotides) for administration to cells in vitro, tocells ex vivo and to cells in vivo, or to a multicellular organism.Particularly preferred in this regard is administration to a humanpatient for treatment of a disease state which is alleviated bydecreasing the level of HIAP3 activity.

[0029] Other objects, features, advantages and aspects of the presentinvention will become apparent to those of skill from the followingdescription. It should be understood, however, that the followingdescription and the specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only.Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following description and from reading the otherparts of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030]FIG. 1 displays the polynucleotide sequence of hiap3, whichencodes HIAP3.

[0031]FIG. 2 displays the deduced amino acid sequence of HIAP3.

[0032]FIG. 3 displays a schematic drawing of the functional domainswithin an HIAP3 polypeptide.

[0033]FIG. 4 demonstrates the amino acid alignment of the BIR domain ofHIAP3 with the BIR domain of other members of the IAP protein family.

[0034]FIG. 5 demonstrates the amino acid alignment of the RING domain ofHIAP3 with the RING domain of other members of the IAP protein family.

[0035] The following drawings depict certain embodiments of theinvention. They are illustrative only and do not limit the inventionotherwise disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

[0036] Definitions

[0037] As used in the specification, examples and appended claims,unless specified to the contrary, the following terms have the meaningindicated.

[0038] “HIAP3” refers to the polypeptide having the amino acid sequenceset out in FIG. 2; variants, analogs, derivatives and fragments thereof,and fragments of the variants, analogs and derivatives. The terms“fragment,” “derivative” and “analog” when referring to the polypeptideof FIG. 2 mean a polypeptide which retains essentially the samebiological activities as the polypeptide of FIG. 2.

[0039] “hiap3” refers to the polynucleotide having the sequence set outin FIG. 1 and polynucleotides encoding polypeptides having the aminoacid sequence of HIAP3 set out in FIG. 2; and to polynucleotidesencoding HIAP3 variants, analogs, derivatives and fragments, andfragments of the variants, analogs and derivatives. hiap3 also refers tosuch polynucleotides composed of RNA as well as to polynucleotides whichare the complement of polynucleotides which encode the polypeptidesequence set out in FIG. 2.

[0040] “Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. Thus, for instance, polynucleotides as used herein refersto, among others, single-and double-stranded DNA, DNA that is a mixtureof single- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, polynucleotide as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.

[0041] As used herein, the term polynucleotide includes DNAs or RNAs asdescribed above that contain one or more modified bases. Thus, DNAs orRNAs with backbones modified for stability or for other reasons are“polynucleotides” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritium-labelled bases, to name just two examples, arepolynucleotides as the term is used herein.

[0042] It will be appreciated that a great variety of modifications havebeen made to DNA and RNA that serve many useful purposes known to thoseof skill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including simple and complex cells,inter alia.

[0043] “Polypeptides”, as used herein, includes all polypeptides asdescribed below. The basic structure of polypeptides is well known andhas been described in innumerable textbooks and other publications inthe art. In this context, the term is used herein to refer to anypeptide or protein comprising two or more amino acids joined to eachother in a linear chain by peptide bonds. As used herein, the termrefers to both short chains, which also commonly are referred to in theart as peptides, oligopeptides and oligomers, for example, and to longerchains, which generally are referred to in the art as proteins, of whichthere are many types.

[0044] It will be appreciated that polypeptides often contain aminoacids other than the 20 amino acids commonly referred to as the 20naturally occurring amino acids, and that many amino acids, includingthe terminal amino acids, may be modified in a given polypeptide, eitherby natural processes such as glycosylation and other post-translationalmodifications, or by chemical modification techniques which are wellknown in the art. Even the common modifications that occur naturally inpolypeptides are too numerous to list exhaustively here, but they arewell described in basic texts and in more detailed monographs, as wellas in a voluminous research literature, and they are well known to thoseof skill in the art. Among the known modifications which may be presentin polypeptides of the present invention are, to name an illustrativefew, acetylation, acylation, ADP-ribosylation, amidation, covalentattachment of flavin, covalent attachment of a heme moiety, covalentattachment of a polynucleotide or polynucleotide derivative, covalentattachment of a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

[0045] Such modifications are well known to those of skill and have beendescribed in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as, for instance, I. E. Creighton, Proteins-Structure and MolecularProperties, 2nd Ed., W. H. Freeman and Company, New York, 1993. Manydetailed reviews are available on this subject, such as, for example,those provided by Wold, F., in Posttranslational Covalent Modificationof Proteins, B. C. Johnson, Ed., Academic Press, New York, pp 1-12,1983; Seifter et al., Meth. Enzymol. 182:626-646, 1990 and Rattan etal., Protein Synthesis: Posttranslational Modifications and Aging, Ann.N.Y. Acad. Sci. 663: 48-62, 1992.

[0046] It will be appreciated, as is well known and as noted above, thatpolypeptides are not always entirely linear. For instance polypeptidesmay be branched as a result of ubiquitination, and they may be circular,with or without branching, generally as a result of Posttranslationalevents, including natural processing events and events brought about byhuman manipulation which do not occur naturally. Circular, branched andbranched circular polypeptides may be synthesized by non-translationalnatural processes and by entirely synthetic methods, as well.

[0047] Modifications can occur anywhere in a polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. In fact, blockage of the amino or carboxyl group in apolypeptide, or both, by a covalent modification, is common in naturallyoccurring and synthetic polypeptides and such modifications may bepresent in polypeptides of the present invention, as well. For instance,the amino terminal residue of polypeptides made in E. coli, prior toproteolytic processing, almost invariably will be N-formylmethionine.

[0048] The modifications that occur in a polypeptide often will be afunction of how it is made. For polypeptides made by expressing a clonedgene in a host, for instance, the nature and extent of the modificationsin large part will be determined by the host cell posttranslationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. For instance, as is well known,glycosylation often does not occur in bacterial hosts such as E. coli.Accordingly, when glycosylation is desired, a polypeptide should beexpressed in a glycosylating host, generally a eukaryotic cell. Insectcells often carry out the same posttranslational glycosylations asmammalian cells and, for this reason, insect cell expression systemshave been developed to efficiently express mammalian proteins havingnative patterns of glycosylation, inter alia. Similar considerationsapply to other modifications.

[0049] It will be appreciated that the same type of modification may bepresent to the same or varying degree at several sites in a givenpolypeptide. Also, a given polypeptide may contain many types ofmodifications.

[0050] In general, as used herein, the term polypeptide encompasses allsuch modifications, particularly those that are present in polypeptidessynthesized by expressing a polynucleotide in a host cell.

[0051] “Polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides which include a sequence encoding apolypeptide of the present invention, particularly the HIAP3 having theamino acid sequence set out in FIG. 2. The term encompassespolynucleotides that include a single continuous region or discontinuousregions encoding the polypeptide (for example, interrupted by introns)together with additional regions.

[0052] “Biological activity” refers to the biologic and/or immunologicactivities of naturally occurring HIAP3.

[0053] “Oligonucleotide(s)” refers to relatively short polynucleotides.Often the term refers to single-stranded deoxyribonucleotides, but itcan refer as well to single- or double-stranded ribonucleotides, RNA:DNAhybrids and double-stranded DNAs, among others. Oligonucleotides, suchas single-stranded DNA probe oligonucleotides, often are synthesized bychemical methods, such as those implemented on automated oligonucleotidesynthesizers. However, oligonucleotides can be made by a variety ofother methods, including in vitro recombinant DNA-mediated techniquesand by expression of DNAs in cells and organisms. “Oligonucleotides” or“oligomers” or polynucleotide “fragment”, “portion”, or “segment” refersto a polynucleotide sequence of at least about 10 nucleotides and asmany as about 60 nucleotides, preferably about 15 to 30 nucleotides, andmore preferably about 20-25 nucleotides.

[0054] “Naturally occurring HIAP3” refers to HIAP3 produced by humancells that have not been genetically engineered and specificallycontemplates various HIAP3 forms arising from post-translationalmodifications of the polypeptide including but not limited toacetylation, carboxylation, glycosylation, phosphorylation, lipidationand acylation.

[0055] “Variant(s)” of polynucleotides or polypeptides, as the term isused herein, are polynucleotides or polypeptides that differ from areference polynucleotide or polypeptide, respectively. Variants in thissense are described below and elsewhere in the present disclosure ingreater detail.

[0056] (1) A polynucleotide that differs in polynucleotide sequence fromanother, reference polynucleotide. Generally, differences are limited sothat the polynucleotide sequences of the reference and the variant areclosely similar overall and, in many regions, identical.

[0057] As noted below, changes in the polynucleotide sequence of thevariant may be silent. That is, they may not alter the amino acidsencoded by the polynucleotide. Where alterations are limited to silentchanges of this type a variant will encode a polypeptide with the sameamino acid sequence as the reference. Also as noted below, changes inthe polynucleotide sequence of the variant may alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide. Suchpolynucleotide changes may result in amino acid substitutions,additions, deletions, fusions and truncations in the polypeptide encodedby the reference sequence, as discussed below.

[0058] (2) A polypeptide that differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference and the variant are closely similaroverall and, in many regions, identical. A variant and referencepolypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions, fusions and truncations, which maybe present in any combination. Recombinant variants encoding these sameor similar polypeptides may be synthesized or selected by making use ofthe “redundancy” in the genetic code. Various codon substitutions, suchas the silent changes which produce various restriction sites, may beintroduced to optimize cloning into a plasmid or viral vector orexpression in a particular prokaryotic or eukaryotic system. Mutationsmay also be introduced to modify the properties of the polypeptide, tochange ligand-binding affinities, interchain affinities, or polypeptidedegradation or turnover rate.

[0059] “Allelic variant” refers to an alternative form of the hiap3polynucleotide. Alleles result from a mutation, i.e., a change in thepolynucleotide sequence, and generally produce altered mRNAs orpolypeptides whose structure or function may or may not be altered. Anygiven gene may have none, one or many allelic forms. Common mutationalchanges which give rise to alleles are generally ascribed to naturaldeletions, additions or substitutions of nucleotides. Each of thesetypes of changes may occur alone, or in combination with the others, orone or more times in a given sequence.

[0060] “Derivative” refers to polynucleotides or polypeptides derivedfrom naturally occurring hiap3 or HIAP3, respectively, by chemicalmodifications such as ubiquitination, labeling (e.g., withradionuclides, various enzymatic modifications), pegylation(derivatization with polyethylene glycol) or by insertion orsubstitution of amino acids such as ornithine (or substitution of thenucleotides which code for such as an amino acid), which do not normallyoccur in human proteins.

[0061] “Deletion” is defined as a change in either polynucleotide oramino acid sequences in which one or more polynucleotides or amino acidresidues, respectively, are absent.

[0062] “Insertion” or “addition” is that change in a polynucleotide oramino acid sequence which has resulted in the addition of one or morepolynucleotides or amino acid residues, respectively, as compared to thenaturally occurring polynucleotide or amino acid sequence.

[0063] “Substitution” results from the replacement of one or morepolynucleotides or amino acids by different polynucleotides or aminoacids, respectively.

[0064] Preferably, amino acid substitutions are the result of replacingone amino acid with another amino acid having similar structural and/orchemical properties, such as the replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, or a threonine witha serine, i.e. conservative amino acid replacement. Insertions ordeletions are typically in the range of about 1 to 5 amino acids. Thevariation allowed may be experimentally determined by systematicallymaking insertions, deletions, or substitutions of amino acids in thepolypeptide using recombinant DNA techniques and assaying the resultingrecombinant variants for activity.

[0065] A polypeptide “fragment”, “portion”, or “segment” is a stretch ofamino acid residues of at least about 5 amino acids, often at leastabout 7 amino acids, typically at least about 9 to 13 amino acids, andin various embodiments, at least about 17 or more amino acids.

[0066] “Recombinant ” or “recombinant DNA molecule” refers to apolynucleotide sequence which is not naturally occurring, or is made bythe artificial combination of two otherwise separated segments ofsequence. By “recombinantly produced” is meant artificial combinationoften accomplished by either chemical synthesis means, or by theartificial manipulation of isolated segments of polynucleotides, e.g.,by genetic engineering techniques. Such manipulation is usually done toreplace a codon with a redundant codon encoding the same or aconservative amino acid, while typically introducing or removing asequence recognition site. Alternatively, it is performed to jointogether polynucleotide segments with desired functions to generate asingle genetic entity comprising a desired combination of functions notfound in the common natural forms. Restriction enzyme recognition sites,regulation sequences, control sequences, or other useful features may beincorporated by design. “Recombinant DNA molecules” include cloning andexpression vectors. “Recombinant” may also refer to a polynucleotidewhich encodes a polypeptide and is prepared using recombinant DNAtechniques.

[0067] “Isolated” means altered “by the hand of man” from its naturalstate; i.e., that, if it occurs in nature, it has been changed orremoved from its original environment, or both. For example, a naturallyoccurring polynucleotide or a polypeptide naturally present in a livinganimal in its natural state is not “isolated”, but the samepolynucleotide or polypeptide separated from the coexisting materials ofits natural state is “isolated”, as the term is employed herein. Forexample, with respect to polynucleotides, the term isolated means thatit is separated from the chromosome and cell in which it naturallyoccurs. Polynucleotides and polypeptides may occur in a composition,such as media formulations, solutions for introduction ofpolynucleotides or polypeptides, for example, into cells, compositionsor solutions for chemical or enzymatic reactions, for instance, whichare not naturally occurring compositions, and, therein remain isolatedpolynucleotides or polypeptides within the meaning of that term as it isemployed herein.

[0068] “Substantially pure” and “substantially homogenous” are usedinterchangeably and describe HIAP3 polypeptide, or fragments thereof, ora DNA segment encoding same, where such polypeptide or DNA molecule isseparated from components that naturally accompany it. An HIAP3polypeptide or fragment thereof, or DNA segment encoding same issubstantially free of naturally-associated components when it isseparated from the native contaminants which accompany it in its naturalstate. Thus, a polypeptide that is chemically synthesized or synthesizedin a cellular system different from the cell in which it naturallyoriginates will be substantially free from its naturally-associatedcomponents. Similarly, a polynucleotide that is chemically synthesizedor synthesized in a cellular system different from the cell in which itnaturally originated will be substantially free from itsnaturally-associated components.

[0069] “Homologous”, when used to describe a polynucleotide, indicatesthat two polynucleotides, or designated sequences thereof, whenoptimally aligned and compared, are identical, with appropriatenucleotide insertions or deletions, in at least 70% of the nucleotides,usually from about 75% to 99%, and more preferably at least about 98 to99% of the nucleotides.

[0070] “Polymerase chain reaction” or “PCR” refers to a procedurewherein specific pieces of DNA are amplified as described in U.S. Pat.No. 4,683,195, issued Jul. 28, 1987. Generally, sequence informationfrom the ends of the polypeptide fragment of interest or beyond needs tobe available, such that oligonucleotide primers can be designed; theseprimers will point towards one another, and will be identical or similarin sequence to opposite strands of the template to be amplified. The 5′terminal nucleotides of the two primers will coincide with the ends ofthe amplified material. PCR can be used to amplify specific DNAsequences from total genomic DNA, cDNA transcribed from total cellularRNA, plasmid sequences, etc. (See generally Mullis et al., Cold SpringHarbor Symp. Quant. Biol., 51: 263, 1987; Erlich, ed., PCR Technology,Stockton Press, NY, 1989).

[0071] “Stringency” typically occurs in a range from about T_(m)(melting temperature)-5° C. (50° below the T_(m) of the probe) to about20° C. to 25° C. below T_(m). As will be understood by those of skill inthe art, a stringency hybridization can be used to identify or detectidentical polynucleotide sequences or to identify or detect similar orrelated polynucleotide sequences. As herein used, the term “stringentconditions” means hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences.

[0072] “Hybridization” as used herein, shall include “any process bywhich a polynucleotide strand joins with a complementary strand throughbase pairing” (Coombs, J., Dictionary of Biotechnology, Stockton Press,New York, N.Y., 1994).

[0073] “Therapeutically effective dose” refers to that amount ofpolypeptide or its antibodies, antagonists, or inhibitors, includingantisense molecules and ribozymes, which ameliorate the symptoms orconditions of a disease state. Therapeutic efficacy and toxicity of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., ED₅₀ (the dosetherapeutically effective in 50% of the population) and LD₅₀ (the doselethal to 50% of the population). The dose ratio between therapeutic andtoxic effects is the therapeutic index, and it can be expressed as theratio, ED₅₀/LD₅₀.

[0074] “Treating” or “treatment” as used herein covers the treatment ofa disease-state in a human patient, which disease-state is associatedwith inappropriate apoptosis, and includes both disease states in whichthe patient is in need of decreased levels of HIAP3 and disease statesin which the patient is in need of increased levels of HIAP3.

DETAILED DESCRIPTION OF THE INVENTION

[0075] The present invention relates to novel HIAP3 polypeptides andhiap3 polynucleotides, among other things, as described in greaterdetail below. In particular, the invention relates to novel HIAP3polypeptides and the polynucleotides encoding these HIAP3 polypeptides,and relates especially to HIAP3 having the amino acid sequence set outin FIG. 2 and hiap3 having the polynucleotide sequence set out inFIG. 1. The present invention also encompasses HIAP3 variants. Apreferred HIAP3 variant is one having at least 70% similarity(preferably at least 70% identity) to the polypeptide sequence shown inFIG. 2 and more preferably at least 90% similarity (more preferably atleast 90% identity) to the polypeptide shown in FIG. 2 and still morepreferably at least 95% similarity (still more preferably at least 95%identity) to the polypeptide sequence shown in FIG. 2 and also includesportions of such polypeptides with such portion of the polypeptidegenerally containing at least 30 amino acids and more preferably atleast 50 amino acids.

[0076] The polynucleotide sequence encoding a portion of HIAP3 was firstidentified as a 226 bp EST (expressed sequence tag; Incyte Clone#1419118) based on analysis of the Incyte EST database using thepolypeptide consensus sequence of the BIR domain of IAPs and the BLASTdatabase search program of the Genetic Computer Group package (OxfordMolecular Group, Campbell, Calif.). The chosen EST sequence demonstrateda 42% identity with the BIR domain of HIAP1 within a stretch of 57 aminoacids. A full length cDNA was obtained by assembling the sequence of theoriginal EST with sequences obtained using 3′ and 5′ RACE (rapidamplification of cDNA end) technology on a fetal kidney cDNA library andthen using the assemble program of the GCG package. Database searching(both public and Incyte databases) identified an Incyte EST (Clone#2953985), containing the 5′ end of the polynucleotide sequence. Thisclone was obtained and sequenced.

[0077] The cDNA sequence designated hiap3 contains 1337 base pairs.There are 169 bp in the 5′ untranslated region and 274 bp in the 3′untranslated region flanking the coding region (see FIG. 1). The codingregion consists of 894 nucelotides with an open reading frame of 298amino acid residues. The initiation codon ATG is located at nucelotideposition 170. Analysis of the deduced polypeptide sequence using theScanProsite feature of the GCG package reveals a single BIR domain fromamino acid position 88 to 155 and a RING zinc finger domain at positions240 to 289.

[0078] The present invention is based in part on the structural homologyshown in FIGS. 4 and 5 for the BIR and RING domains among HIAP3 andother members of the IAP protein family. There is approximately a 50%identity of the BIR domain between HIAP3 and the third BIR domain ofeither human or mouse IAP1 or IAP2. There is a 74% identity of the RINGdomain between HIAP3 and HIAP1. A Pile Up analysis shows very highhomology within the members of the family as shown in FIGS. 4 and 5.Northern blot analysis indicated that the polynucleotide encoding HIAP3is expressed mainly in placenta, lymph node, fetal kidney, and kidneytumor, giving it a tissue pattern distribution different from all otherknown human IAPs.

[0079] Polynucleotides

[0080] In accordance with one aspect of the present invention, there areprovided isolated polynucleotides that encode the HIAP3 polypeptidehaving the deduced amino acid sequence of FIG. 2.

[0081] Using the information provided herein, such as the polynucleotidesequence set out in FIG. 1, a polynucleotide of the present inventionencoding an HIAP3 polypeptide may be obtained using standard cloning andscreening procedures, such as those for cloning cDNAs using mRNA fromcells of human tissue as starting material. Illustrative of theinvention, the polynucleotide sequence in FIG. 1 was found in a humanfetal kidney cDNA library.

[0082] Polynucleotides of the present invention may be in the form ofRNA, such as mRNA, or in the form of DNA, including, for instance, cDNAand genomic DNA obtained by cloning or produced by chemical synthetictechniques or by a combination thereof. The DNA may be double-strandedor single-stranded. Single-stranded DNA may be the coding strand, alsoknown as the sense strand, or it may be the non-coding strand, alsoreferred to as the anti-sense strand.

[0083] The coding sequence which encodes the polypeptide may beidentical to the coding sequence of the polynucleotide shown in FIG. 1.It also may be a polynucleotide with a different sequence, which, as aresult of the redundancy (degeneracy) of the genetic code, encodes thepolypeptide of FIG. 2.

[0084] Polynucleotides of the present invention which encode thepolypeptide of FIG. 2 may include, but are not limited to the codingsequence for the mature polypeptide, by itself; the coding sequence forthe mature polypeptide and additional coding sequences, such as thoseencoding a leader or secretory sequence, such as a pre-, or pro- orprepro-protein sequence; the coding sequence of the mature polypeptide,with or without the aforementioned additional coding sequences, togetherwith additional, non-coding sequences, including for example, but notlimited to introns and non-coding 5′ and 3′ sequences, such as thetranscribed, non-translated sequences that play a role in transcription,mRNA processing—including splicing and polyadenylation signals, forexample—ribosome binding and stability of mRNA; additional codingsequence which codes for additional amino acids, such as those whichprovide additional functionalities. Thus, for instance, the polypeptidemay be fused to a marker sequence, such as a peptide, which facilitatespurification of the fused polypeptide. In certain preferred embodimentsof this aspect of the invention, the marker sequence is a hexa-histidinepeptide, such as the tag provided in a pcDNA3.1Myc-His vector(Invitrogen, Carlsbad, Calif.) among others, many of which arecommercially available. As described in Gentz et al. (Proc. Natl. Acad.Sci., USA 86: 821-824, 1989), for instance, hexa-histidine provides forconvenient purification of the fusion protein. The HA tag corresponds toan epitope derived from influenza hemagglutinin protein, which has beendescribed by Wilson et al. (Cell 37: 767, 1984), for instance.

[0085] The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 2. A variant of the polynucleotide may be a naturally occurringvariant such as a naturally occurring allelic variant, or it may be avariant that is not known to occur naturally. Such non-naturallyoccurring variants of the polynucleotide may be made by mutagenesistechniques, including those applied to polynucleotides, cells ororganisms.

[0086] Among variants in this regard are variants that differ from theaforementioned polynucleotides by polynucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more polynucleotides. The variants may be altered incoding or non-coding regions or both. Alterations in the coding regionsmay produce conservative or non-conservative amino acid substitutions,deletions or additions.

[0087] Among the particularly preferred embodiments of the invention inthis regard are polynucleotides encoding polypeptides having the aminoacid sequence of HIAP3 set out in FIG. 2; variants, analogs, derivativesand fragments thereof, and fragments of the variants, analogs andderivatives.

[0088] Further particularly preferred in this regard are polynucleotidesencoding HIAP3 variants, analogs, derivatives and fragments, andvariants, analogs and derivatives of the fragments, which have the aminoacid sequence of the HIAP3 polypeptide of FIG. 2 in which several, afew, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues aresubstituted, deleted or added, in any combination. Especially preferredamong these are silent substitutions, additions and deletions, which donot alter the properties and activities of the HIAP3 polypeptide. Alsoespecially preferred in this regard are conservative substitutions. Mosthighly preferred are polynucleotides encoding polypeptides having theamino acid sequence of FIG. 2 without substitutions.

[0089] Further preferred embodiments of the invention arepolynucleotides that are at least 70% identical to a polynucleotideencoding the HIAP3 polypeptide having the amino acid sequence set out inFIG. 2, and polynucleotides which are complementary to suchpolynucleotides. Alternatively, most highly preferred arepolynucleotides that comprise a region that is at least 80% identical toa polynucleotide encoding the HIAP3 polypeptide and polynucleotidescomplementary thereto. In this regard, polynucleotides at least 90%identical to the same are particularly preferred, and among theseparticularly preferred polynucleotides, those with at least 95% areespecially preferred. Furthermore, those with at least 97% are highlypreferred among those with at least 95%, and among these, those with atleast 98% and at least 99% are particularly highly preferred with atleast 99% being the more preferred.

[0090] Particularly preferred embodiments in this respect, moreover, arepolynucleotides which encode polypeptides which retain substantially thesame biological activity as the mature polypeptide encoded by thepolynucleotide sequence of FIG. 1.

[0091] The present invention further relates to polynucleotides thathybridize to the herein above-described sequences. In this regard, thepresent invention especially relates to polynucleotides which hybridizeunder stringent conditions to the herein above-describedpolynucleotides. As herein used, the term “stringent conditions” meanshybridization will occur only if there is at least 95% and preferably atleast 97% identity between the sequences.

[0092] As discussed additionally herein regarding polynucleotide assaysof the invention, for instance, polynucleotides of the invention asdiscussed above, may be used as a hybridization probes for cDNA andgenomic DNA to isolate full-length cDNAs and genomic clones encodingHIAP3 and to isolate cDNA and genomic clones of other genes that have ahigh sequence similarity to the hiap3 gene. Such probes generally willcomprise at least 15 bases. Preferably, such probes will have at least30 bases and may have at least 50 bases. Particularly preferred probeswill have at least 30 bases and will have 50 bases or less.

[0093] For example, the coding region of the hiap3 gene may be isolatedby screening using the known DNA sequence to synthesize anoligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a polypeptide of the present invention is thenused to screen a library of human cDNA, genomic DNA or mRNA to determineto which members of the library the probe hybridizes.

[0094] The polynucleotides and polypeptides of the present invention maybe employed as research reagents and materials for discovery oftreatments and diagnostics to human disease as further discussed hereinrelating to polynucleotide assays, inter alia.

[0095] The polynucleotides may encode a polypeptide which is the matureprotein plus additional amino or carboxyl-terminal amino acids, or aminoacids interior to the mature polypeptide (when the mature form has morethan one polypeptide chain, for instance). Such sequences may play arole in processing of a protein from precursor to a mature form, mayfacilitate protein trafficking, may prolong or shorten protein half-lifeor may facilitate manipulation of a protein for assay or production,among other things. As generally is the case in situ, the additionalamino acids may be processed away from the mature protein by cellularenzymes.

[0096] A precursor protein, having the mature form of the polypeptidefused to one or more prosequences may be an inactive form of thepolypeptide. When prosequences are removed such inactive precursorsgenerally are activated. Some or all of the prosequences may be removedbefore activation. Generally, such precursors are called proproteins.

[0097] In sum, a polynucleotide of the present invention may encode amature protein, a mature protein plus a leader sequence (which may bereferred to as a preprotein), a precursor of a mature protein having oneor more prosequences which are not the leader sequences of a preprotein,or a preproprotein, which is a precursor to a proprotein, having aleader sequence and one or more prosequences, which generally areremoved during processing steps that produce active and mature forms ofthe polypeptide.

[0098] Polypeptides

[0099] The present invention further relates to an HIAP3 polypeptidewhich has the deduced amino acid sequence of FIG. 2.

[0100] The invention also relates to fragments, analogs and derivativesof these polypeptides. The terms fragment, derivative and analog whenreferring to the polypeptide of FIG. 2 means a polypeptide which retainsessentially the same biological activity as such a polypeptide. Thus, ananalog includes a proprotein which can be activated by cleavage of theproprotein portion to produce an active mature polypeptide.

[0101] The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide. Incertain preferred embodiments it is a recombinant polypeptide.

[0102] The fragment, derivative or analog of the polypeptide of FIG. 2may be (i) one in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol) or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asa leader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

[0103] Among the particularly preferred embodiments of the invention inthis regard are polypeptides having the amino acid sequence of HIAP3 setout in FIG. 2, variants, analogs, derivatives and fragments thereof, andvariants, analogs and derivatives of the fragments. Alternatively,particularly preferred embodiments of the invention in this regard arepolypeptides having the amino acid sequence of the HIAP3 shown in FIG.2, variants, analogs, derivatives and fragments thereof, and variants,analogs and derivatives of the fragments.

[0104] Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and lle, interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe and Tyr.

[0105] Further particularly preferred in this regard are variants,analogs, derivatives and fragments, and variants, analogs andderivatives of the fragments, having the amino acid sequence of theHIAP3 polypeptide of FIG. 2 in which several, a few, 5 to 10, 1 to 5, 1to 3, 2, 1 or no amino acid residues are substituted, deleted or added,in any combination. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the HIAP3. Also especially preferred inthis regard are conservative substitutions. Most highly preferred arepolypeptides having the amino acid sequence of FIG. 2 withoutsubstitutions.

[0106] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

[0107] The polypeptides of the present invention also include thepolypeptide of FIG. 2 (in particular the mature polypeptide) as well aspolypeptides which have at least 70% similarity (preferably at least 70%identity) to the polypeptide of FIG. 2 and more preferably at least 90%similarity (more preferably at least 90% identity) to the polypeptide ofFIG. 2 and still more preferably at least 95% similarity (still morepreferably at least 95% identity) to the polypeptide of FIG. 2 and alsoinclude portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

[0108] As known in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide.

[0109] Fragments or portions of the polypeptides of the presentinvention may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.Fragments or portions of the polynucleotides of the present inventionmay be used to synthesize full-length polynucleotides of the presentinvention.

[0110] Fragments

[0111] Also among preferred embodiments of this aspect of the presentinvention are polypeptides comprising fragments of HIAP3, mostparticularly fragments of the HIAP3 having the amino acid sequence setout in FIG. 2, and fragments of variants and derivatives of the HIAP3 ofFIG. 2.

[0112] In this regard a fragment is a polypeptide having an amino acidsequence that entirely is the same as part but not all of the amino acidsequence of the aforementioned HIAP3 polypeptides and variants orderivatives thereof.

[0113] Such fragments may be “free-standing,” i.e., not part of or fusedto other amino acids or polypeptides, or they may be comprised within alarger polypeptide of which they form a part or region. When comprisedwithin a larger polypeptide, the presently discussed fragments mostpreferably form a single continuous region. However, several fragmentsmay be comprised within a single larger polypeptide. For instance,certain preferred embodiments relate to a fragment of an HIAP3polypeptide of the present invention comprised within a precursorpolypeptide designed for expression in a host and having heterologouspre- and propolypeptide regions fused to the amino terminus of the HIAP3fragment and an additional region fused to the carboxyl terminus of thefragment. Therefore, fragments in one aspect of the meaning intendedherein, refers to the portion or portions of a fusion polypeptide orfusion protein derived from HIAP3.

[0114] As representative examples of polypeptide fragments of theinvention, there may be mentioned those which have from about 25 toabout 145 amino acids.

[0115] In this context about includes the particularly recited range andranges larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acidat either extreme or at both extremes. For instance, about 145 aminoacids in this context means a polypeptide fragment of 25 plus or minusseveral, a few, 5, 4, 3, 2 or 1 amino acids to 145 plus or minus severala few, 5, 4, 3, 2 or 1 amino acid residues, i.e., ranges as broad as 25minus several amino acids to 145 plus several amino acids to as narrowas 25 plus several amino acids to 145 minus several amino acids.

[0116] Highly preferred in this regard are the recited ranges plus orminus as many as 5 amino acids at either or at both extremes.Particularly highly preferred are the recited ranges plus or minus asmany as 3 amino acids at either or at both the recited extremes.Especially particularly highly preferred are ranges plus or minus 1amino acid at either or at both extremes or the recited ranges with noadditions or deletions. Most highly preferred of all in this regard arefragments from about 25 to about 145 amino acids.

[0117] Among especially preferred fragments of the invention aretruncation mutants of HIAP3. Truncation mutants include HIAP3polypeptides having the amino acid sequence of FIG. 2, or of variants orderivatives thereof, except for deletion of a continuous series ofresidues (that is, a continuous region, part or portion) that includesthe amino terminus, or a continuous series of residues that includes thecarboxyl terminus or, as in double truncation mutants, deletion of twocontinuous series of residues, one including the amino terminus and oneincluding the carboxyl terminus. Fragments having the size ranges setout above also are preferred embodiments of truncation fragments, whichare especially preferred among fragments generally.

[0118] Also preferred in this aspect of the invention are fragmentscharacterized by structural or functional attributes of HIAP3. Mostpreferred are fragments containing the BIR and C-terminal RING zincfinger domains of HIAP3.

[0119] Certain preferred regions in these regards are set out in FIG. 3,and include, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence set out in FIG. 2. Asset out in FIG. 3, such preferred regions include the BIR domain and theC-terminal RING zinc finger domain.

[0120] Among highly preferred fragments in this regard are those thatcomprise regions of HIAP3 that combine several structural features, suchas several of the features set out above. In this regard, the BIR andRING domains defined by the amino acid residues from about 88-155 andfrom about 240-289, respectively, of FIG. 2, which are characteristic ofthe IAP family of proteins are especially highly preferred regions. Suchregions may be comprised within a larger polypeptide or may be bythemselves a preferred fragment of the present invention, as discussedabove. It will be appreciated that the term “about” as used in thisparagraph has the meaning set out above regarding fragments in general.

[0121] Further preferred regions are those that mediate activities ofHIAP3. Most highly preferred in this regard are fragments that have achemical, biological or other activity of HIAP3, including those with asimilar activity or an improved activity, or with a decreasedundesirable activity. Highly preferred in this regard are fragments thatcontain regions that are homologs in sequence, or in position, or inboth sequence and position to active regions of related polypeptides,such as the other proteins of the IAP family, which includes HIAP3.

[0122] It will be appreciated that the invention also relates to, amongothers, polynucleotides encoding the aforementioned fragments,polynucleotides that hybridize to polynucleotides encoding thefragments, particularly those that hybridize under stringent conditions,and polynucleotides, such as PCR primers, for amplifying polynucleotidesthat encode the fragments. In these regards, preferred polynucleotidesare those that correspond to the preferred fragments, as discussedabove.

[0123] Vectors, Host Cells, and Expression Systems

[0124] The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques. Suchtechniques are described in Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., 1989 andAusubel, F. M. et al., Current Protocols in Molecular Biology, JohnWiley & Sons, New York, N.Y., 1989.

[0125] Host cells can be genetically engineered to incorporatepolynucleotides and express polypeptides of the present invention. Forinstance, polynucleotides may be introduced into host cells using wellknown techniques of infection, transduction, transfection, transvectionand transformation. The polynucleotides may be introduced alone or withother polynucleotides. Such other polynucleotides may be introducedindependently, co-introduced or introduced joined to the polynucleotidesof the invention.

[0126] Thus, for instance, polynucleotides of the invention may betransfected into host cells with another, separate, polynucleotideencoding a selectable marker, using standard techniques forco-transfection and selection in, for instance, mammalian cells. In thiscase, the polynucleotides generally will be stably incorporated into thehost cell genome.

[0127] Alternatively, the polynucleotides may be joined to a vectorcontaining a selectable marker for propagation in a host. The vectorconstruct may be introduced into host cells by the aforementionedtechniques. Generally, a plasmid vector is introduced as DNA in aprecipitate, such as a calcium phosphate precipitate, or in a complexwith a charged lipid. Electroporation also may be used to introducepolynucleotides into a host. If the vector is a virus, it may bepackaged in vitro or introduced into a packaging cell and the packagedvirus may be transduced into cells. A wide variety of techniquessuitable for making polynucleotides and for introducing polynucleotidesinto cells in accordance with this aspect of the invention are wellknown and routine to those of skill in the art. Such techniques arereviewed at length in Sambrook et al. cited above, which is illustrativeof the many laboratory manuals that detail these techniques. Inaccordance with this aspect of the invention, the vector may be, forexample, a plasmid vector, a single or double-stranded phage vector, asingle or double-stranded RNA or DNA viral vector. Such vectors may beintroduced into cells as polynucleotides, preferably DNA, by well knowntechniques for introducing DNA and RNA into cells. The vectors, in thecase of phage and viral vectors, also may be and preferably areintroduced into cells as packaged or encapsidated virus by well knowntechniques for infection and transduction. Viral vectors may bereplication competent or replication defective. In the latter case viralpropagation generally will occur only in complementing host cells.

[0128] Preferred among vectors, in certain respects, are those forexpression of polynucleotides and polypeptides of the present invention.Generally, such vectors comprise cis-acting control regions effectivefor expression in a host operatively linked to the polynucleotide to beexpressed. Appropriate trans-acting factors either are supplied by thehost, supplied by a complementing vector or supplied by the vectoritself upon introduction into the host.

[0129] In certain preferred embodiments in this regard, the vectorsprovide for specific expression. Such specific expression may beinducible expression or expression only in certain types of cells orboth inducible and cell-specific. Particularly preferred among induciblevectors are vectors that can be induced for expression by environmentalfactors that are easy to manipulate, such as temperature and nutrientadditives. A variety of vectors suitable to this aspect of theinvention, including constitutive and inducible expression vectors foruse in prokaryotic and eukaryotic hosts, are well known and employedroutinely by those of skill in the art.

[0130] The engineered host cells can be cultured in conventionalnutrient media, which may be modified as appropriate for, inter alia,activating promoters, selecting transformants or amplifying genes.Culture conditions, such as temperature, pH and the like, previouslyused with the host cell selected for expression generally will besuitable for expression of polypeptides of the present invention as willbe apparent to those of skill in the art.

[0131] A great variety of expression vectors can be used to express apolypeptide of the invention. Such vectors include chromosomal, episomaland virus-derived vectors e.g., vectors derived from bacterial plasmids,from bacteriophage, from yeast episomes, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV4O, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids, all may be used for expression inaccordance with this aspect of the present invention. Generally, anyvector suitable to maintain, propagate or express polynucleotides toexpress a polypeptide in a host may be used for expression in thisregard.

[0132] The appropriate DNA sequence may be inserted into the vector byany of a variety of well-known and routine techniques. In general, a DNAsequence for expression is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionendonucleases and then joining the restriction fragments together usingT4 DNA ligase. Procedures for restriction and ligation that can be usedto this end are well known and routine to those of skill. Suitableprocedures in this regard, and for constructing expression vectors usingalternative techniques, which also are well known and routine to thoseof skill, are set forth in great detail in Sambrook et al. citedelsewhere herein.

[0133] The DNA sequence in the expression vector is operatively linkedto appropriate expression control sequence(s), including, for instance,a promoter to direct mRNA transcription. Representatives of suchpromoters include the phage lambda PL promoter, the E. coli lac, trp andtac promoters, the SV4O early and late promoters and promoters ofretroviral LTRs, to name just a few of the well-known promoters. It willbe understood that numerous promoters not mentioned are suitable for usein this aspect of the invention, are well known and may readily beemployed by those of skill in the manner illustrated by the discussionand the examples herein.

[0134] In general, expression constructs will contain sites fortranscription initiation and termination and, in the transcribed region,a ribosome binding site for translation. The coding portion of themature transcripts expressed by the constructs will include atranslation initiating AUG at the beginning and a termination codonappropriately positioned at the end of the polypeptide to be translated.

[0135] In addition, the constructs may contain control regions thatregulate as well as engender expression. Generally, in accordance withmany commonly practiced procedures, such regions will operate bycontrolling transcription, such as repressor binding sites andenhancers, among others.

[0136] Vectors for propagation and expression generally will includeselectable markers. Such markers also may be suitable for amplificationor the vectors may contain additional markers for this purpose. In thisregard, the expression vectors preferably contain one or more selectablemarker genes to provide a phenotypic trait for selection of transformedhost cells. Preferred markers include dihydrofolate reductase orneomycin resistance for eukaryotic cell culture, and tetracycline,theomycin, kanamycin or ampicillin resistance genes for culturing E.coli and other bacteria.

[0137] The vector containing the appropriate DNA sequence as describedelsewhere herein, as well as an appropriate promoter, and otherappropriate control sequences, may be introduced into an appropriatehost using a variety of well known techniques suitable to expressiontherein of a desired polypeptide. Representative examples of appropriatehosts include bacterial cells, such as E. coli. Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila 52 and Spodoptera Sf9 cells; animal cells suchas CHO, COS and Bowes melanoma cells; and plant cells. Hosts for a greatvariety of expression constructs are well known, and those of skill willbe enabled by the present disclosure readily to select a host forexpressing a polypeptides in accordance with this aspect of the presentinvention.

[0138] Various mammalian cell culture systems can be employed forexpression, as well. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblast (Gluzman et al., Cell 23:175, 1991). Other cell lines capable of expressing a compatible vectorinclude for example, the C127, 3T3, CHO, HeLa, human kidney 293 and BHKcell lines. In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the polynucleotide sequence coding for HIAP3 may beligated into an adenovirus transcription/translation complex consistingof the late promoter and tripartite leader sequence. Insertion in anonessential E1 or E3 region of the viral genome will result in a viablevirus capable of expressing HIAP3 in infected host cells (Logan andShenk, Proc. Natl. Acad. Sci. USA 81:3655-59, 1984). In addition,transcription enhancers, such as the rouse sarcoma virus (RVS) enhancer,may be used to increase expression in mammalian host cells.

[0139] More particularly, the present invention also includesrecombinant constructs, such as expression constructs, comprising one ormore of the sequences described above. The constructs comprise a vector,such as a plasmid or viral vector, into which such a sequence of theinvention has been inserted. The sequence may be inserted in a forwardor reverse orientation. In certain preferred embodiments in this regard,the construct further comprises regulatory sequences, including, forexample, a promoter, operably linked to the sequence. Large numbers ofsuitable vectors and promoters are known to those of skill in the art,and there are many commercially available vectors suitable for use inthe present invention.

[0140] The following vectors, which are commercially available, areprovided by way of example. Among vectors preferred for use in bacteriaare pQE7O, pQE6O and pQE-9, available from Qiagen USA (Valencia,Calif.); pBS vectors, Phagescript® vectors, Bluescript® vectors, pNH8A,pNHI6a, pNHI8A, pNH46A, available from Stratagene (LaJolla, Calif.); andptrc99a, pK223-3, pKK233-3, pDR54O, pRIT5 available from PharmaciaBiotech (Piscataway, N.J.). Most preferred is the pGEX-6P-3 vector,available from Pharmacia Biotech. Among preferred eukaryotic vectors arepWLNEO, pSV2CAT, pOG44, PXTI and pSG available from Stratagene; andPSVK3, pBPV, pMSG and pSVL available from Pharmacia Biotech. Mostpreferred are the vectors pcDNA3.1 Myc-His and pRc/CMV2 available fromIntrogene. These vectors are listed solely by way of illustration of themany commercially available and well known vectors that are available tothose of skill in the art for use in accordance with this aspect of thepresent invention. It will be appreciated that any other plasmid orvector suitable for, for example, introduction, maintenance, propagationor expression of a polynucleotide or polypeptide of the invention in ahost may be used in this aspect of the invention.

[0141] Promoter regions can be selected from any desired gene usingvectors that contain a reporter transcription unit lacking a promoterregion, such as a chloramphenicol acetyl transferase (“cat”)transcription unit, downstream of restriction site or sites forintroducing a candidate promoter fragment; i.e., a fragment that maycontain a promoter. As is well known, introduction into the vector of apromoter-containing fragment at the restriction site upstream of the catgene engenders production of CAT activity, which can be detected bystandard CAT assays. Vectors suitable to this end are well known andreadily available. Two such vectors are pKK232-B and pCM7. Thus,promoters for expression of polynucleotides of the present inventioninclude not only well known and readily available promoters, but alsopromoters that readily may be obtained by the foregoing technique, usinga reporter gene.

[0142] Among known bacterial promoters suitable for expression ofpolynucleotides and polypeptides in accordance with the presentinvention are the E. coli lacI and lacZ promoters, the T3 and T7promoters, the T5 tac promoter, the lambda PR, PL promoters and the trppromoter. Among known eukaryotic promoters suitable in this regard arethe CMV immediate early promoter, the HSV thymidine kinase promoter, theearly and late SV4O promoters, the promoters of retroviral LTRs, such asthose of the Rous sarcoma virus (“RSV”) and metallothionein promoters,such as the mouse metallothionein- I promoter.

[0143] Selection of appropriate vectors and promoters for expression ina host cell is a well known procedure and the requisite techniques forexpression vector construction, introduction of the vector into the hostand expression in the host are routine skills in the art.

[0144] Generally, recombinant expression vectors will include origins ofreplication, a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence, and a selectablemarker to permit isolation of vector containing cells after exposure tothe vector.

[0145] The present invention also relates to host cells containing theabove-described constructs discussed above. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Constructs in host cells can be used in aconventional manner to produce the gene product encoded by therecombinant sequence. Alternatively, the polypeptides of the presentinvention can be produced by direct peptide synthesis using solid-phasetechniques (Stewart et al., Solid-Phase Peptide Synthesis, W. H. FreemanCo., San Francisco, 1969; Merrifield, J., J. Am. Chem. Soc.85:2149-2154, 1963). In vitro protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may be achieved,for example, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer, Foster City, Calif.) in accordance with the instructions providedby the manufacturer. Various fragments of HIAP3 may be chemicallysynthesized separately and combined using chemical methods to producethe full length molecule.

[0146] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook et al., citedelsewhere herein.

[0147] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act to increase transcriptionalactivity of a promoter in a given host cell-type. Examples of enhancersinclude the SV4O enhancer, which is located on the late side of thereplication origin at bp 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0148] Polynucleotides of the invention, encoding the heterologousstructural sequence of a polypeptide of the invention generally will beinserted into the vector using standard techniques so that it isoperably linked to the promoter for expression. The polynucleotide willbe positioned so that the transcription start site is locatedappropriately 5′ to a ribosome binding site. The ribosome binding sitewill be 5′ to the AUG that initiates translation of the polypeptide tobe expressed. Generally, there will be no other open reading frames thatbegin with an initiation codon, usually AUG, and lie between theribosome binding site and the initiating AUG. Also, generally, therewill be a translation stop codon at the end of the polypeptide and therewill be a polyadenylation signal and a transcription termination signalappropriately disposed at the 3′ end of the transcribed region.

[0149] For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals. Thepolypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals but also additionalheterologous fuctional regions. Thus for instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, special regions also may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to polypeptides to engender secretion or excretion, to improvestability and to facilitate purification, among others, are familiar androutine techniques in the art. For example, when large quantities ofHIAP3 are needed for the induction of antibodies, vectors which directhigh level expression of fusion proteins that are readily purified maybe desirable. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBluescript® (Stratagene), in which the hiap3 coding sequence may beligated into the vector in frame with sequence for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heede and Shuster, J. Biol. Chem.264:5503-5509, 1989) and the like. PGEX vectors (Promega, Madison, Wis.)may also be used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems are designed to includeheparin, thrombin or factor Xa protease cleavage sites so that thecloned polypeptide of interest can be released from the GST moiety atwill.

[0150] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, where the selectedpromoter is inducible it is induced by appropriate means (e.g.,temperature shift or exposure to chemical inducer) and cells arecultured for an additional period.

[0151] Cells typically then are harvested by centrifugation, disruptedby physical or chemical means, and the resulting crude extract retainedfor further purification.

[0152] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, suchmethods are well know to those skilled in the art.

[0153] The HIAP3 polypeptide can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.Well known techniques for refolding protein may be employed toregenerate active conformation when the polypeptide is denatured duringisolation and or purification. Various other methods of proteinpurification well known in the art include those described in Deutscher,M., Methods in Enzymology, Vol 182, Academic Press, San Diego, 1982; andScopes, R., Protein Purification: Principles and PracticeSpringer-Verlag, New York, 1982.

[0154] Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

[0155] Uses of HIAP3 Polypeptides and the Polynucleotides which EncodeThem

[0156] hiap3 polynucleotides and HIAP3 polypeptides may be used inaccordance with the present invention for a variety of applications,particularly those that make use of the chemical and biologicalproperties of HIAP3. Additional applications relate to diagnosis and totreatment of disorders of cells, tissues and organisms related to thepresence of inappropriate apoptosis. These aspects of the invention areillustrated further by the following discussion and are describedfurther within the body of the specification.

[0157] The rationale for the use of the polynucleotide and polypeptidesequences of the present invention is based in part on the chemical andstructural homology between the HIAP3 disclosed herein and other IAPmolecules. HIAP3 may be used in the diagnosis and treatment ofconditions, disorders or diseases associated with abnormal orinappropriate apoptosis. These would include, but are not limited to,cancer and chronic viral infections, in which apoptosis is insufficient,and neurodegenerative disorders and chronic heart failure, which arecharacterized by premature apoptosis.

[0158] hiap3 polynucleotide sequences can be used for chromosomeidentification and as DNA probes.

[0159] HIAP3 polypeptides can be used to generate antibodies to HIAP3which may be useful in detecting the levels of HIAP3 protein in cellsand tissues.

[0160] Polynucleotides encoding HIAP3 may be useful in diagnostic assaysfor detecting the levels of polynucleotides encoding HIAP3 in cells andtissues.

[0161] In conditions associated with overexpression of HIAP3, such ascancer, it may be advantageous to suppress HIAP3 production, thusreducing HIAP3 levels. HIAP3 could be suppressed by administration ofantisense oligonucleotides or ribozymes. Alternatively, antibodiesspecifically recognizing areas of the HIAP3 polypeptide which areresponsible for its activity may be introduced to treat diseases orconditions associated with abnormal HIAP3 activity.

[0162] In conditions associated with underexpression of HIAP3, such asneurodegenerative disorders, it may be advantageous to increase HIAP3levels. HIAP3 could be supplied to the patient using gene therapy, inwhich expression of HIAP3 polypeptide occurs in vivo within the patientfollowing administration of suitable recombinant molecules containingpolynucleotides encoding and expressing HIAP3 polypeptide.

[0163] Polynucleotide Assays

[0164] This invention is also related to the use of the HIAP3polynucleotides to detect complementary polynucleotides such as, forexample, as a diagnostic reagent. Detection of a mutated form of HIAP3associated with a dysfunction will provide a diagnostic tool that canadd or define a diagnosis of a disease or susceptibility to a diseasewhich results from under-expression, over-expression or alteredexpression of HIAP3, such as, for example, neoplasia andneurodegenerative disorders.

[0165] Individuals carrying mutations in the gene encoding HIAP3 may bedetected at the DNA level by a variety of techniques. Polynucleotidesamples for diagnosis may be obtained from a patient's cells, such asfrom blood, urine, saliva, tissue biopsy and autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR prior to analysis (Saiki et al., Nature, 324:163-166, 1986). RNA or cDNA may also be used in the same ways. As anexample, PCR primers complementary to the polynucleotide sequenceencoding HIAP3 can be used to identify and analyze hiap3 expression andmutations. For example, deletions and insertions can be detected by achange in size of the amplified product in comparison to the normalgenotype. Point mutations can be identified by hybridizing amplified DNAto radiolabeled hiap3 RNA or alternatively, radiolabeled hiap3 antisenseDNA sequences. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase A digestion or by differences in meltingtemperatures.

[0166] Sequence differences between a reference gene and genes havingmutations also may be revealed by direct DNA sequencing. In addition,cloned DNA segments may be employed as probes to detect specific DNAsegments. The sensitivity of such methods can be greatly enhanced byappropriate use of PCR or another amplification method. For example, asequencing primer is used with double-stranded PCR product or asingle-stranded template molecule generated by a modified PCR. Thesequence determination is performed by conventional procedures withradiolabeled polynucleotide or by automatic sequencing procedures withfluorescent tags.

[0167] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels, with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230: 1242, 1985).

[0168] Sequence changes at specific locations also may be revealed bynuclease protection assays, such as RNase and S1 protection or thechemical cleavage method (e.g., Catton et al., Proc. Natl. Acad. Sci.,USA, 85:4397-4401, 1985).

[0169] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,restriction fragment length polymorphisms (“RFLP”) and Southern blottingof genomic DNA).

[0170] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations also can be detected by in situ analysis.

[0171] Chromosome Assays

[0172] The polynucleotide sequences of the present invention are alsovaluable for chromosome identification. The sequence is specificallytargeted to and can hybridize with a particular location on anindividual human chromosome. Moreover, there is a current need foridentifying particular sites on the chromosome. Few chromosome markingreagents based on actual sequence data (repeat polymorphisms) arepresently available for marking chromosomal location. The mapping ofDNAs to chromosomes according to the present invention is an importantfirst step in correlating those sequences with genes associated withdisease.

[0173] In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of an hiap3 gene. This can beaccomplished using a variety of well known techniques and libraries,which generally are available commercially. The genomic DNA then is usedfor in situ chromosome mapping using well known techniques for thispurpose. Typically, in accordance with routine procedures for chromosomemapping, some trial and error may be necessary to identify a genomicprobe that gives a good in situ hybridization signal.

[0174] In some cases, in addition, sequences can be mapped tochromosomes by preparing PCR primers (preferably 15-25 bp) from thecDNA. Computer analysis of the 3′ untranslated region of the gene isused to rapidly select primers that do not span more than one exon inthe genomic DNA, thus complicating the amplification process. Theseprimers are then used for PCR screening of somatic cell hybridscontaining individual human chromosomes. Only those hybrids containingthe human gene corresponding to the primer will yield an amplifiedfragment.

[0175] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0176] Fluorescence in situ hybridization (“FISH”) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60 bases. For a review of this technique, see Verma etal., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York, 1988.

[0177] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man, available on line throughJohns Hopkins University, Welch Medical Library. The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0178] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0179] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb). In situ hybridization of chromosomal preparations andphysical mapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. For example,an STS based map of the human genome was published by the Whitehead-MITCenter for Genomic Research (Hudson et al., Science 270:1945-1954,1995). Often the placement of a gene on the chromosome of anothermammalian species such as mouse (Whitehead Institute/MIT Center forGenome Research, Genetic Map of the Mouse, Database Release 10, Apr. 28,1995) may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncea disease or syndrome, such as ataxia telangiectasia (AT) has beencrudely localized by genetic linkage to a particular genomic region, forexample, AT to 11q22-23 (Gatti et al., Nature 336:577-580, 1988), anysequences mapping to that area may represent associated or regulatorygenes for further investigation. The nucleotide sequence of the presentinvention may also be used to detect differences in the chromosomallocation due to translocation, inversion, etc. among normal, carrier oraffected individuals.

[0180] Polypeptide Assays

[0181] The present invention also relates to a diagnostic assays such asquantitative and diagnostic assays for detecting levels of HIAP3 proteinin cells and tissues, including determination of normal and abnormallevels. Thus, for instance, a diagnostic assay in accordance with theinvention for detecting over-expression of HIAP3 protein compared tonormal control tissue samples may be used to detect the presence ofneoplasia, for example, tumors. Assay techniques that can be used todetermine levels of a protein, such as an HIAP3 protein of the presentinvention, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays (RIA),competitive-binding assays, western Blot analysis and enzyme-linkedimmunoabsorbant assays (ELISA), and fluorescent activated cell sorting(FACS). Among these ELISAs frequently are preferred. An ELISA assayinitially comprises preparing an antibody specific to HIAP3, preferablya monoclonal antibody. In addition a reporter antibody generally isprepared which binds to the monoclonal antibody. The reporter antibodyis attached to a detectable reagent such as a radioactive, fluorescentor enzymatic reagent, in this example horseradish peroxidase enzyme.

[0182] To carry out an ELISA a sample is removed from a host andincubated on a solid support, e.g. a polystyrene dish, that binds theproteins in the sample. Any free protein binding sites on the dish arethen covered by incubating with a non-specific protein such as bovineserum albumin. Next, the monoclonal antibody is incubated in the dishduring which time the monoclonal antibodies attach to any HIAP3 proteinsattached to the polystyrene dish. Unbound monoclonal antibody is washedout with buffer. The reporter antibody linked to horseradish peroxidaseis placed in the dish resulting in binding of the reporter antibody toany monoclonal antibody bound to HIAP3. Unattached reporter antibody isthen washed out. Reagents for peroxidase activity, including acolorimetric substrate are then added to the dish. Immobilizedperoxidase, linked to HIAP3 through the primary and secondaryantibodies, produces a colored reaction product. The amount of colordeveloped in a given time period indicates the amount of HIAP3 proteinpresent in the sample. Quantitative results typically are obtained byreference to a standard curve.

[0183] A competition assay may be employed wherein antibodies specificto HIAP3 are attached to a solid support and labeled HIAP3 and a samplederived from the host are passed over the solid support and the amountof label detected attached to the solid support can be correlated to aquantity of HIAP3 in the sample.

[0184] These and other assays are described, among other places, inHampton et al. (Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn., 1990) and Maddox et al. (J. Exp. Med. 158:12111, 1983).

[0185] Antibodies

[0186] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as an immunogen toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

[0187] Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

[0188] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler and Milstein,Nature 256: 495-497, 1975), the human B-cell hybridoma technique (Kozboret al., Immunology Today 4:72, 1983) and the EBV-hybridoma technique toproduce human monoclonal antibodies (Cole et al., in MonoclonalAntibodies and Cancer, Alan R. Liss, Inc., 77-96, 1985).

[0189] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison et al., Proc. Natl. Acad. Sci.USA 81:6851-6855, 1984; Neuberger et al., Nature 312:604-608, 1984;Takeda et al., Nature 314:452-454, 1985). Alternatively, techniquesdescribed for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce HIAP3-specific single chainantibodies.

[0190] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inOrlandi et al. (Proc. Natl. Acad. Sci. USA 86:3833-3837, 1989) andWinter and Milstein (Nature 349:293-299, 1991).

[0191] Antibody fragments which contain specific binding sites for HIAP3may also be generated. For example, such fragments include, but are notlimited to the F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse et al., Science 256:1270-1281, 1989).

[0192] The above-described antibodies may be employed to isolate or toidentify clones expressing the polypeptide or purify the polypeptide ofthe present invention by attachment of the antibody to a solid supportfor isolation and/or purification by affinity chromatography.

[0193] Pharmaceutical Compositions and Administration

[0194] The present invention also relates to pharmaceutical compositionswhich may comprise hiap3 polynucleotides, HIAP3 proteins, antibodies,agonists, antagonists, or inhibitors, alone or in combination with atleast one other agent, such as stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. Any of these molecules can be administered to a patient alone, orin combination with other agents, drugs or hormones, in pharmaceuticalcompositions where it is mixed with excipient(s) or pharmaceuticallyacceptable carriers. In one embodiment of the present invention, thepharmaceutically acceptable carrier is pharmaceutically inert.

[0195] The present invention also relates to the administration ofpharmaceutical compositions. Such administration is accomplished orallyor parenterally. Methods of parenteral delivery include topical,intra-arterial (directly to the tumor), intramuscular, subcutaneous,intramedullary, intrathecal, intraventricular, intravenous,intraperitoneal, or intranasal administration. In addition to the activeingredients, these pharmaceutical compositions may contain suitablepharmaceutically acceptable carriers comprising excipients andauxilliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Further details ontechniques for formulation and administration may be found in the latestedition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co,Easton, Pa.).

[0196] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions and thelike, for ingestion by the patient.

[0197] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxilliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potatoe, or other plants; cellulose suchas methyl, cellulose, hydroxypropylmethylcellulose, or sodiumcarboxymethylcellulose; and gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0198] Dragee cores are provided with suitable coatings such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, ie. dosage.

[0199] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders such aslactose or starches, lubricants such as talc or magnesium stearate, andoptionally, stabilizers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

[0200] Pharmaceutical formulations for parenteral administration includeaqueous solutions of active compounds. For injection, the pharmaceuticalcompositions of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hank'ssolution, Ringer's solution, or physiologically buffered saline. Aqueousinjection suspensions may contain substances which increase viscosity ofthe suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0201] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0202] Kits

[0203] The invention further relates to pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, reflecting approval by theagency of the manufacture, use or sale of the product for humanadministration.

[0204] Manufacture and Storage

[0205] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

[0206] The pharmaceutical composition may be provided as a salt and canbe formed with may acids, including by not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents that are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose,2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with bufferprior to use.

[0207] After pharmaceutical compositions comprising a compound of theinvention formulated in an acceptable carrier have been prepared, theycan be placed in an appropriate container and labeled for treatment ofan indicated condition. For administration of HIAP3, such labeling wouldinclude amount, frequency and method of administration.

[0208] Therapeutically Effective Dose

[0209] Pharmaceutical compositions suitable for use in the presentinvention include compositions wherein the active ingredients arecontained in an effective amount to achieve the intended purpose, i.e.treatment of a particular disease state characterized by inappropriateapoptosis. The determination of an effective dose is well within thecapability of those skilled in the art.

[0210] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model is also used to achieve a desirable concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

[0211] A therapeutically effective dose refers to that amount of proteinor its antibodies, antagonists, or inhibitors which ameliorate thesymptoms or condition. Therapeutic efficacy and toxicity of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., ED₅₀ (the dosetherapeutically effective in 50% of the population) and LD₅₀ (the doselethal to 50% of the population). The dose ratio between therapeutic andtoxic effects is the therapeutic index, and it can be expressed as theratio, ED₅₀/LD₅₀. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies is used in formulating a range of dosage forhuman use. The dosage of such compounds lies preferably within a rangeof circulating concentrations what include the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0212] The exact dosage is chosen by the individual physician in view ofthe patient to be treated. Dosage and administration are adjusted toprovide sufficient levels of the active moiety or to maintain thedesired effect. Additional factors which may be taken into accountinclude the severity of the disease state, eg, tumor size and location;age, weight and gender of the patient; diet, time and frequency ofadministration, drug combination(s), reaction sensitivities, andtolerance/response to therapy. Long acting pharmaceutical compositionsmight be administered every 3 to 4 days, every week, or once every twoweeks depending on half-life and clearance rate of the particularformulation.

[0213] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature. See U.S. Pat. Nos. 4,657,760;5,206,344; or 5,225,212. Those skilled in the art will employ differentformulations for polynucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0214] Gene Therapy

[0215] The hiap3 polynucleotides and HIAP3 polypeptides of the presentinvention may be employed in accordance with the present invention byexpression of such polypeptides in vivo, in treatment modalities oftenreferred to as “gene therapy.”

[0216] Thus, for example, cells from a patient may be engineered with apolynucleotide, such as a DNA or RNA, encoding a polypeptide ex vivo,and the engineered cells then can be provided to a patient to be treatedwith the polypeptide. For example, cells may be engineered ex vivo bythe use of a retroviral plasmid vector containing RNA encoding apolypeptide of the present invention. Such methods are well-known in theart and their use in the present invention will be apparent from theteachings herein.

[0217] Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by procedures known in the art. For example, apolynucleotide of the invention may be engineered for expression in areplication defective retroviral vector, as discussed above. Theretroviral expression construct then may be isolated and introduced intoa packaging cell is transduced with a retroviral plasmid vectorcontaining RNA encoding a polypeptide of the present invention such thatthe packaging cell now produces infectious viral particles containingthe gene of interest. These producer cells may be administered to apatient for engineering cells in vivo and expression of the polypeptidein vivo. These and other methods for administering a polypeptide of thepresent invention by such method should be apparent to those skilled inthe art from the teachings of the present invention.

[0218] Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, MoloneyMurine Leukemia Virus, spleen necrosis virus, retroviruses such as RousSarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon apeleukemia virus, human immunodeficiency virus, adenovirus,Myeloproliferative Sarcoma Virus, and mammary tumor virus. In oneembodiment, the retroviral plasmid vector is derived from Moloney MurineLeukemia Virus.

[0219] Such vectors will include one or more promoters for expressingthe polypeptide. Suitable promoters which may be employed include, butare not limited to, the retroviral LTR; the SV4O promoter; and the humancytomegalovirus (CMV) promoter (Miller et al., Biotechniques 7:980-990,1989), or any other promoter (e.g., cellular promoters such aseukaryotic cellular promoters including, but not limited to, thehistone, RNA polymerase III, and, β actin promoters). Other viralpromoters which may be employed include, but are not limited to,adenovirus promoters, thymidine kinase (TK) promoters, and B19parvovirus promoters. The selection of a suitable promoter will beapparent to those skilled in the art from the teachings containedherein.

[0220] The polynucleotide sequence encoding the polypeptide of thepresent invention will be placed under the control of a suitablepromoter. Suitable promoters which may be employed include, but are notlimited to, adenoviral promoters, such as the adenoviral major latepromoter; or heterologous promoters, such as the cytomegalovirus CMV)promoter; the respiratory syncytial virus (RSV) promoter; induciblepromoters, such as the MMT promoter, the metallothionein promoter; heatshock promoters; the albumin promoter; the ApoAl promoter; human globinpromoters; viral thymidine kinase promoters such as the Herpes Simplexthymidine kinase promoter; retroviral LTRs (including the modifiedretroviral LTRs herein above described); the β-actin promoter; and humangrowth hormone promoters. The promoter also may be the native promoterwhich controls the gene encoding the polypeptide.

[0221] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE5OI,PA317 Y-2, Y-AM, PAI2, T19-14X, VT-19-17-H2, YCRE, YCRIP, GP+E-86,GP+envAml2, and DAN cell lines as described in Miller, A., Human GeneTherapy 1: 5-14, 1990. The vector may be transduced into the packagingcells through any means known in the art. Such means include, but arenot limited to, electroporation, the use of liposomes, and CaPO₄precipitation. In one alternative, the retroviral plasmid vector may beencapsulated into a liposome, or coupled to a lipid, and thenadministered to a host.

[0222] The producer cell line will generate infectious retroviral vectorparticles, which include the polynucleotide sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the polynucleotide(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

[0223] Therapeutic Use of Antisense Vectors and Ribozymes

[0224] Expression vectors derived from retroviruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids, may be also beused for construction and delivery of recombinant vectors which willexpress anti-sense hiap3. See, for example, the techniques described inSambrook et al. (supra) and Ausubel et al. (supra).

[0225] The polynucleotides comprising full length cDNA sequence and/orits regulatory elements enable researchers to use hiap3 polynucleotidesas an investigative tool in sense (Youssoufian and Lodish, Mol. Cell.Biol. 13:98-104, 1993) or antisense (Eguchi, et al., Annu. Rev. Biochem.60:631-652, 1991) regulation of gene function. Such technology is nowwell known in the art, and sense or antisense oligomers, or largerfragments, can be designed from various locations along the coding orcontrol regions.

[0226] Genes encoding HIAP3 can be turned off by transfecting a cell ortissue with expression vectors which express high levels of a desiredHIAP3 fragment. Such constructs can flood cells with untranslatablesense or antisense sequences. Even in the absence of integration intothe DNA, such vectors may continue to transcribe RNA molecules until allcopies are disabled by endogenous nucleases. Transient expression maylast for a month or more with a non-replicating vector and even longerif appropriate replication elements are part of the vector system.

[0227] As mentioned above, modification of gene expression can beobtained by designing antisense molecules, DNA or RNA, to controlregions of hiap3, ie, the promoters, enhancers, and introns.Oligonucleotides derived from the transcription initiation site, eg,between −10 and +10 regions of the leader sequence, are preferred. Theantisense molecules may also be designed to block translation of mRNA bypreventing the transcript from binding to ribosomes. Similarly,inhibition can be achieved using “triple helix” base-pairingmethodology. Triple helix pairing compromises the ability of the doublehelix to open sufficiently for the binding of polymerases, transcriptionfactors, or regulatory molecules. Recent therapeutic advances usingtriplex DNA were reviewed by Gee, J. E. et al. (In Huber and Car,Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco,N.Y., 1994).

[0228] Ribozymes are enzymatic RNA molecules capable of catalyzing thespecific cleavage of RNA (U.S. Pat. No. 4,987,071; WO 93/23057). Themechanism of ribozyme action involves sequence-specific hybridization ofthe ribozyme molecule to complementary target RNA, followed byendonucleolytic cleavage. Within the scope of the invention areengineered hammerhead motif ribozyme molecules that can specifically andefficiently catalyze endonucleolytic cleavage of RNA encoding HIAP3.

[0229] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences, GUA, GUU and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays (Irie et al., Advance. Pharmacol.40:207-257, 1997).

[0230] Antisense molecules and ribozymes of the invention may beprepared by any method known in the art for the synthesis of RNAmolecules. These include techniques for chemically synthesizingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription or DNA sequences encoding HIAP3. Such DNA sequences may beincorporated into a wide variety of vectors which suitable RNApolymerase promoters such as T7 or SP6. Alternatively, antisense cDNAconstructs that synthesize antisense RNA constitutively or inducibly canbe introduced into cell lines, cells or tissues.

[0231] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecules or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule.Increased stability can also be achieved by the inclusion ofnontraditional bases such as inosine and queosine as well as acetyl-,methyl-, thio- and similarly modified forms of adenine, cytidine,guanine, thymine, and uridine which are not as easily recognized byendogenous endonucleases.

[0232] Methods for introducing vectors into cells or tissues includethose methods discussed infra and which are equally suitable for invivo, in vitro and ex vivo therapy. For ex vivo therapy, vectors areintroduced into stem cells taken from the patient and clonallypropagated for autologous transplant back into that same patient aspresented in U.S. Pat. Nos. 5,399,493 and 5,437,994, disclosed herein byreference. Delivery by transfection and by liposome are quite will knownin the art.

EXAMPLES

[0233] The present invention is further described by the followingexamples. The examples are provided solely to illustrate the inventionby reference to specific embodiments. These exemplifications, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

[0234] All examples were carried out using standard techniques, whichare well known and routine to those of skill in the art, except whereotherwise described in detail. Routine molecular biology techniques ofthe following examples can be carried out as described in standardlaboratory manuals, such as Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

[0235] 1. Isolation of Human iap3 cDNA Clone

[0236] A 226 bp EST sequence (Incyte Clone #1419118) was identifiedbased on analysis of the Incyte EST database using the polypeptideconsensus sequence of the BIR domain of IAP. tblastn database (GCGpackage) searches using the peptide sequence predicted from the ESTsequence revealed a significant degree of similarity between amino acidresidues 19 and 75 of this gene product and the partial BIR domain ofhuman inhibitor of apoptosis protein 1(HIAP1). There is a 42% to 47%identity between this 57 amino acid gene product and partial BIR1, BIR2and BIR3 sequences of HIAP1, suggesting that the gene product is ahomolog of HIAP1.

[0237] A. 5′ and 3′ RACE (5′ and 3′ Rapid Amplification of cDNA End)

[0238] Oligonucleotide primers derived from the EST sequence was used toclone 5′-end and 3′-end cDNA. Marathon-ready human fetal kidney cDNA waspurchased from Clontech Inc. (South San Francisco) and used as template.RACE reactions were carried out according to the manufacturer'sinstructions (Clontech Inc).

[0239] Oligonucleotide primers used in 5′RACE are:5′CCTTCCTGGCTCCTGGGCACTTTCAGA3′ 5′GCCCCCATAGCAGAAGAAGCACCTC3′5′GACGCAACTCCTCAGAGCCCATGCC3′

[0240] Oligonucleotide primers used in 3′RACE are5′GGCATGGGCTCTGAGGAGTTG3′ 5′CAAGGTGAGGTGCTTCTTCTGCTA3′

[0241] The PCR products were cloned into pCRII vector (Invitrogen). Atotal of 12 clones were sequenced. 7 clones have sequences whichoverlapped with the EST sequence.

[0242] B. DNA and Protein Sequence Analysis

[0243] The DNA sequences of the above clones and EST clone wereassembled using GCG assemble program. The resulting contiguous DNAsequence was used to search databases. Two clones were identified inIncyte database and one clone from public EST database. One clone(2953985) was ordered from Incyte and sequenced. Sequencing data showedthat the cDNA sequence contains 1337 bp. This cDNA sequence isdesignated hiap3. There is 169 bp in the 5′ untranslated region and 274bp in the 3′ untranslated region flanking the coding region (FIGS. 1 and3).

[0244] The coding region consists of 894 bp. An open reading frame of298 amino acid residues was identified. The initiation codon ATG islocated at polynucleotide position 170, which has an adequate Kozaksequence with an upstream stop codon at polynucleotide position 110(FIGS. 1 and 3).

[0245] Analysis of the predicted protein sequence (FIG. 2) reveals asingle BIR domain from amino acid position 88 to 155 (FIG. 4). There isapproximately 50% identity of the BIR domain between HIAP3 and the thirdBIR domain of either human or mouse IAP1 or IAP2. Analysis using theGenetic Computer Group PileUp algorithm indicates very high homologywith other members of the IAP family (FIGS. 4 & 5). HIAP3 has the BIRsignature residues including three cysteines and a histidine. A RINGdomain containing a peptide sequence from aa position 240 to 289 wasalso identified, which has the signature cysteine, leucine and histidineresidues (FIG. 4). There is 84% identity of the RING domain betweenHIAP3 and human HIAP-1. A PileUp analysis shows very high homologywithin the members of the family (FIG. 5)

[0246] 2. Northern Blot Analysis

[0247] A 414 bp Ava1 fragment, isolated from a 5′RACE clone (#1-1L2generated using primer 5′GCCCCCATAGCAGAAGAAGCACCTC3′) was used to probeNorthern blots. This fragment was labeled with ³²P-dCTP using high primelabeling kit purchased from Boehringer Mannheim. Human Multiple TissueNorthern Blot I and II, Human Fetal Tissue Blot, Human Immune SystemTissue Blot (Clontech) and Human Multiple Tumor Tissue Blot (Biochain)were probed at high stringency following the supplier's instructions(Clontech). The results indicates that hiap3 is expressed mainly inplacenta, lymph node, fetal kidney and kidney tumor. These experimentswere confirmed using a second probe, a 387 bp Narl-Kpnl fragmentisolated from a second 5′RACE clone (#9-9-1, generated using primer5′CCTTCCTGGCTCCTGGGCACTTTCAGA3′).

[0248] Analysis of the Incyte database using the BLAST algorithm fromthe GCG package with the full length cDNA sequence of hiap3 generated atotal of 9 EST clones (including the original EST clone) in the Incytedatabase. These clones are 2953985, 1419118, 1418580, 3842242, 4589046,1529102, 3673370, 1520835 and EST92646. Three clones are from fetalkidney library, one clone from mast cell library, one clone fromdendritic cell library, one clone from mononuclear cell derived fromumbilical vein, one clone from placenta, one clone from bladder tumor,and one clone from skin tumor tissue. The distribution of these ESTclones is similar to that found in the Northern blot analysis andsuggests that HIAP3 may play a role in development, hematopoietic/immunesystem and tumor formation.

[0249] All publications and patents mentioned in the above specificationare herein incorporated by reference. While the present invention hasbeen described with reference to the specific embodiments thereof, itshould be understood by those skilled in the art that various changesmay be made and equivalents may be substituted without departing fromthe true spirit and scope of the invention. In addition, manymodifications may be made to adapt a particular situation, material,composition of matter, process, process step or steps, to the objective,spirit and scope of the present invention. All such modifications areintended to be within the scope of the claims appended hereto.

1 7 1 1337 DNA Homo sapiens CDS (170)..(1066) 1 gggatactcc cctcccagggtgtctggtgg caggcctgtg cctatccctg ctgtccccag 60 ggtgggcccc gggggtcaggagctccagaa gggccagctg ggcatattct gagattggcc 120 atcagccccc atttctgctgcaaacctggt cagagccagt gttccctcc atg gga cct 178 Met Gly Pro 1 aaa gacagt gcc aag tgc ctg cac cgt gga cca cag ccg agc cac tgg 226 Lys Asp SerAla Lys Cys Leu His Arg Gly Pro Gln Pro Ser His Trp 5 10 15 gca gcc ggtgat ggt ccc acg cag gag cgc tgt gga ccc cgc tct ctg 274 Ala Ala Gly AspGly Pro Thr Gln Glu Arg Cys Gly Pro Arg Ser Leu 20 25 30 35 ggc agc cctgtc cta ggc ctg gac acc tgc aga gcc tgg gac cac gtg 322 Gly Ser Pro ValLeu Gly Leu Asp Thr Cys Arg Ala Trp Asp His Val 40 45 50 gat ggg cag atcctg ggc cag ctg cgg ccc ctg aca gag gag gaa gag 370 Asp Gly Gln Ile LeuGly Gln Leu Arg Pro Leu Thr Glu Glu Glu Glu 55 60 65 gag gag ggc gcc ggggcc acc ttg tcc agg ggg cct gcc ttc ccc ggc 418 Glu Glu Gly Ala Gly AlaThr Leu Ser Arg Gly Pro Ala Phe Pro Gly 70 75 80 atg ggc tct gag gag ttgcgt ctg gcc tcc ttc tat gac tgg ccg ctg 466 Met Gly Ser Glu Glu Leu ArgLeu Ala Ser Phe Tyr Asp Trp Pro Leu 85 90 95 act gct gag gtg cca ccc gagctg ctg gct gct gcc ggc ttc ttc cac 514 Thr Ala Glu Val Pro Pro Glu LeuLeu Ala Ala Ala Gly Phe Phe His 100 105 110 115 aca ggc cat cag gac aaggtg agg tgc ttc ttc tgc tat ggg ggc ctg 562 Thr Gly His Gln Asp Lys ValArg Cys Phe Phe Cys Tyr Gly Gly Leu 120 125 130 cag agc tgg aag cgc ggggac gac ccc tgg acg gag cat gcc aag tgg 610 Gln Ser Trp Lys Arg Gly AspAsp Pro Trp Thr Glu His Ala Lys Trp 135 140 145 ttc ccc agc tgt cag ttcctg ctc cgg tca aaa gga aga gac ttt gtc 658 Phe Pro Ser Cys Gln Phe LeuLeu Arg Ser Lys Gly Arg Asp Phe Val 150 155 160 cac agt gtg cag gag actcac tcc cag ctg ctg ggc tct tgg gac ccg 706 His Ser Val Gln Glu Thr HisSer Gln Leu Leu Gly Ser Trp Asp Pro 165 170 175 tgg gaa gaa ccg gaa gacgca gcc cct gtg gcc ccc tcc gtc cct gcc 754 Trp Glu Glu Pro Glu Asp AlaAla Pro Val Ala Pro Ser Val Pro Ala 180 185 190 195 tct ggg tac cct gagctg ccc aca ccc agg aga gag gtc cag tct gaa 802 Ser Gly Tyr Pro Glu LeuPro Thr Pro Arg Arg Glu Val Gln Ser Glu 200 205 210 agt gcc cag gag ccagga ggg gtc agt cca gcc gag gcc cag agg gcg 850 Ser Ala Gln Glu Pro GlyGly Val Ser Pro Ala Glu Ala Gln Arg Ala 215 220 225 tgg tgg gtt ctt gagccc cca gga gcc agg gat gtg gag gcg cag ctg 898 Trp Trp Val Leu Glu ProPro Gly Ala Arg Asp Val Glu Ala Gln Leu 230 235 240 cgg cgg ctg cag gaggag agg acg tgc aag gtg tgc ctg gac cgc gcc 946 Arg Arg Leu Gln Glu GluArg Thr Cys Lys Val Cys Leu Asp Arg Ala 245 250 255 gtg tcc atc gtc tttgtg ccg tgc ggc cac ctg gtc tgt gct gag tgt 994 Val Ser Ile Val Phe ValPro Cys Gly His Leu Val Cys Ala Glu Cys 260 265 270 275 gcc ccc ggc ctgcag ctg tgc ccc atc tgc aga gcc ccc gtc cgc agc 1042 Ala Pro Gly Leu GlnLeu Cys Pro Ile Cys Arg Ala Pro Val Arg Ser 280 285 290 cgc gtg cgc accttc ctg tcc tag gccaggtgcc atggccggcc aggtgggctg 1096 Arg Val Arg ThrPhe Leu Ser 295 cagagtgggc tccctgcccc tctctgcctg ttctggactg tgttctgggcctgctgagga 1156 tggcagagct ggtgtccatc cagcactgac cagccctgat tccccgaccaccgcccaggg 1216 tggagaagga ggcccttgct tggcgtgggg gatggcttaa ctgtacctgtttggatgctt 1276 ctgaatagaa ataaagtggg ttttccctgg aggtacccag caaaaaaaaaaaaaaaaaaa 1336 a 1337 2 298 PRT Homo sapiens 2 Met Gly Pro Lys Asp SerAla Lys Cys Leu His Arg Gly Pro Gln Pro 1 5 10 15 Ser His Trp Ala AlaGly Asp Gly Pro Thr Gln Glu Arg Cys Gly Pro 20 25 30 Arg Ser Leu Gly SerPro Val Leu Gly Leu Asp Thr Cys Arg Ala Trp 35 40 45 Asp His Val Asp GlyGln Ile Leu Gly Gln Leu Arg Pro Leu Thr Glu 50 55 60 Glu Glu Glu Glu GluGly Ala Gly Ala Thr Leu Ser Arg Gly Pro Ala 65 70 75 80 Phe Pro Gly MetGly Ser Glu Glu Leu Arg Leu Ala Ser Phe Tyr Asp 85 90 95 Trp Pro Leu ThrAla Glu Val Pro Pro Glu Leu Leu Ala Ala Ala Gly 100 105 110 Phe Phe HisThr Gly His Gln Asp Lys Val Arg Cys Phe Phe Cys Tyr 115 120 125 Gly GlyLeu Gln Ser Trp Lys Arg Gly Asp Asp Pro Trp Thr Glu His 130 135 140 AlaLys Trp Phe Pro Ser Cys Gln Phe Leu Leu Arg Ser Lys Gly Arg 145 150 155160 Asp Phe Val His Ser Val Gln Glu Thr His Ser Gln Leu Leu Gly Ser 165170 175 Trp Asp Pro Trp Glu Glu Pro Glu Asp Ala Ala Pro Val Ala Pro Ser180 185 190 Val Pro Ala Ser Gly Tyr Pro Glu Leu Pro Thr Pro Arg Arg GluVal 195 200 205 Gln Ser Glu Ser Ala Gln Glu Pro Gly Gly Val Ser Pro AlaGlu Ala 210 215 220 Gln Arg Ala Trp Trp Val Leu Glu Pro Pro Gly Ala ArgAsp Val Glu 225 230 235 240 Ala Gln Leu Arg Arg Leu Gln Glu Glu Arg ThrCys Lys Val Cys Leu 245 250 255 Asp Arg Ala Val Ser Ile Val Phe Val ProCys Gly His Leu Val Cys 260 265 270 Ala Glu Cys Ala Pro Gly Leu Gln LeuCys Pro Ile Cys Arg Ala Pro 275 280 285 Val Arg Ser Arg Val Arg Thr PheLeu Ser 290 295 3 27 DNA artificial sequence race primer 1 3 ccttcctggctcctgggcac tttcaga 27 4 25 DNA artificial sequence race primer 2 4gcccccatag cagaagaagc acctc 25 5 25 DNA artificial sequence race primer3 5 gacgcaactc ctcagagccc atgcc 25 6 21 DNA artificial sequence raceprimer 4 6 ggcatgggct ctgaggagtt g 21 7 24 DNA artificial sequence raceprimer 5 7 caaggtgagg tgcttcttct gcta 24

What is claimed is:
 1. An isolated polynucleotide comprising a polynucleotide having at least a 70% identity to a member selected from the group consisting of: (a) a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in FIG. 2; (b) a polynucleotide encoding a polypeptide comprising amino acid 88 to amino acid 289 as set forth in FIG. 2; (c) a polynucleotide encoding a polypeptide comprising amino acid 88 to amino acid 155 as set forth in FIG. 2; (d) a polynucleotide encoding a polypeptide comprising amino acid 240 to amino acid 289 as set forth in FIG. 2; and (e) polynucleotide which is complementary to the polynucleotide of (a), (b), (c) or (d).
 2. The polynucleotide of claim 1 wherein the polynucleotide is DNA.
 3. The polynucleotide of claim 1 wherein the polynucleotide is RNA.
 4. The polynucleotide of claim 1 wherein the polynucleotide is genomic DNA.
 5. The polynucleotide of claim 2 wherein the polynucleotide encodes the polypeptide comprising amino acids 1 to 298 as set forth in FIG.
 2. 6. The polynucleotide of claim 2 wherein the polynucleotide encodes the polypeptide comprising amino acid 88 to amino acid 289 as set forth in FIG.
 2. 7. The polynucleotide of claim 2 wherein the polynucleotide encodes the polypeptide comprising amino acid 88 to amino acid 155 as set forth in FIG.
 2. 8. The polynucleotide of claim 2 wherein the polynucleotide encodes the polypeptide comprising amino acid 240 to amino acid 289 as set forth in FIG.
 2. 9. The polynucleotide of claim 1 wherein the polynucleotide comprises the sequence as set forth in FIG. 1 from nucleotide 1 to nucleotide
 1337. 10. The polynucleotide of claim 1 wherein the polynucleotide comprises the sequence as set forth in FIG. 1 from nucleotide 170 to nucleotide
 1064. 11. An vector comprising the polynucleotide of claim
 2. 12. A host cell comprising the vector of claim
 11. 13. A method of producing a polypeptide comprising expressing from the host cell of claim 12 the polypeptide encoded by the polynucleotide.
 14. The method of claim 13 wherein the polypeptide comprises amino acid 1 to amino acid 298 as set forth in FIG.
 2. 15. The method of claim 13 wherein the polypeptide comprises amino acid 88 to amino acid 289 as set forth in FIG.
 2. 16. The method of claim 13 wherein the polypeptide comprises amino acid 88 to amino acid 155 as set forth in FIG.
 2. 17. The method of claim 13 wherein the polypeptide comprises amino acid 240 to amino acid 289 as set forth in FIG.
 2. 18. A method of producing a polypeptide wherein the polypeptide comprises the amino acid sequence shown in FIG. 2, the method comprising the steps of: (a) culturing the host cell of claim 12 under conditions whereby the polypeptide is expressed; and (b) recovering the polypeptide from the culture.
 19. A method for producing a cell which expresses a polypeptide comprising genetically engineering the cell with the vector of claim
 11. 20. A polypeptide comprising a member selected from the group consisting of: (a) a polypeptide having the amino acid sequence as set forth in FIG. 2; (b) a polypeptide comprising amino acid 88 to amino acid 289 as set forth in FIG. 2; (c) a polypeptide comprising amino acid 88 to amino acid 155 as set forth in FIG. 2; (d) a polypeptide comprising amino acid 240 to amino acid 289 as set forth in FIG. 2; and (e) a polypeptide which is at least 70% identical to the polypeptide of (a), (b), (c) or (d).
 21. The polypeptide of claim 20 wherein the polypeptide comprises amino acid 1 to amino acid 298 as set forth in FIG.
 2. 21. The polypeptide of claim 20 wherein the polypeptide comprises amino acid 88 to amino acid 289 as set forth in FIG.
 2. 22. The polypeptide of claim 20 wherein the polypeptide comprises amino acid 88 to amino acid 155 as set forth in FIG.
 2. 23. The polypeptide of claim 20 wherein the polypeptide comprises amino acid 240 to amino acid 289 as set forth in FIG. 2
 24. A method of treating a disease-state in a human patient which disease-state is associated with inappropriate apoptosis and wherein the patient is in need of increased levels of a polypeptide comprising a member selected from the group consisting of: (a) a polypeptide having the amino acid sequence as set forth in FIG. 2; (b) a polypeptide comprising amino acid 88 to amino acid 289 as set forth in FIG. 2; (c) a polypeptide comprising amino acid 88 to amino acid 155 as set forth in FIG. 2; (d) a polypeptide comprising amino acid 240 to amino acid 289 as set forth in FIG. 2; and (e) a polypeptide which is at least 70% identical to the polypeptide of (a), (b), (c) or (d); wherein the method comprises administering to the patient a therapeutically effective amount of the polypeptide.
 25. The method of claim 24 wherein said therapeutically effective amount of the polypeptide is administered by providing to the patient a polynucleotide encoding the polypeptide and expressing the polypeptide in vivo.
 26. A method of treating a disease-state in a human patient which disease-state is associated with inappropriate apoptosis and wherein the patient is in need of decreased levels of a polypeptide comprising a member selected from the group consisting of: (a) a polypeptide having the amino acid sequence as set forth in FIG. 2; (b) a polypeptide comprising amino acid 88 to amino acid 289 as set forth in FIG. 2; (c) a polypeptide comprising amino acid 88 to amino acid 155 as set forth in FIG. 2; (d) a polypeptide comprising amino acid 240 to amino acid 289 as set forth in FIG. 2; and (e) a polypeptide which is at least 70% identical to the polypeptide of (a), (b), (c) or (d); wherein the method comprises administering to the patient a therapeutically effective amount of a ribozyme which specifically cleaves RNA encoding the polypeptide.
 27. A method of treating a disease-state in a human patient which disease-state is associated with inappropriate apoptosis and wherein the patient is in need of decreased levels of a polypeptide comprising a member selected from the group consisting of: (a) a polypeptide having the amino acid sequence as set forth in FIG. 2; (b) a polypeptide comprising amino acid 88 to amino acid 289 as set forth in FIG. 2; (c) a polypeptide comprising amino acid 88 to amino acid 155 as set forth in FIG. 2; (d) a polypeptide comprising amino acid 240 to amino acid 289 as set forth in FIG. 2; and (e) a polypeptide which is at least 70% identical to the polypeptide of (a), (b), (c) or (d); awherein the method comprises administering to the patient a therapeutically effective amount of a polynucleotide which is complementary to a polynucleotide encoding the polypeptide.
 28. A diagnostic method wherein the method comprises analyzing for the presence of a polypeptide comprising a member selected from the group consisting of: (a) a polypeptide having the amino acid sequence as set forth in FIG. 2; (b) a polypeptide comprising amino acid 88 to amino acid 289 as set forth in FIG. 2; (c) a polypeptide comprising amino acid 88 to amino acid 155 as set forth in FIG. 2; (d) a polypeptide comprising amino acid 240 to amino acid 289 as set forth in FIG. 2; and (e) a polypeptide which is at least 70% identical to the polypeptide of (a), (b), (c) or (d); in a sample derived from a host.
 29. A diagnostic method wherein the method comprises analyzing for the presence of a polynucleotide comprising a polynucleotide having at least 70% identity to a member selected from the group consisting of: (a) a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in FIG. 2; (b) a polynucleotide encoding a polypeptide comprising amino acid 88 to amino acid 289 as set forth in FIG. 2; (c) a polynucleotide encoding a polypeptide comprising amino acid 88 to amino acid 155 as set forth in FIG. 2; (d) a polynucleotide encoding a polypeptide comprising amino acid 240 to amino acid 289 as set forth in FIG. 2; and (e) polynucleotide which is complementary to the polynucleotide of (a), (b), (c) or (d); in a sample derived from a host.
 30. An isolated antibody which is specific for a polypeptide comprising a member selected from the group consisting of: (a) a polypeptide having the amino acid sequence as set forth in FIG. 2; (b) a polypeptide comprising amino acid 88 to amino acid 289 as set forth in FIG. 2; (c) a polypeptide comprising amino acid 88 to amino acid 155 as set forth in FIG. 2; (d) a polypeptide comprising amino acid 240 to amino acid 289 as set forth in FIG. 2; and (e) a polypeptide which is at least 70% identical to the polypeptide of (a), (b), (c) or (d). 